Isolated human kinase proteins, nucleic acid molecules encoding human kinase proteins, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the kinase peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the kinase peptides, and methods of identifying modulators of the kinase peptides.

FIELD OF THE INVENTION

[0001] The present invention is in the field of kinase proteins that arerelated to the serine/threonine protein kinase subfamily, recombinantDNA molecules, and protein production. The present inventionspecifically provides novel peptides and proteins that effect proteinphosphorylation and nucleic acid molecules encoding such peptide andprotein molecules, all of which are useful in the development of humantherapeutics and diagnostic compositions and methods.

BACKGROUND OF THE INVENTION

[0002] Protein Kinases

[0003] Kinases regulate many different cell proliferation,differentiation, and signaling processes by adding phosphate groups toproteins. Uncontrolled signaling has been implicated in a variety ofdisease conditions including inflammation, cancer, arteriosclerosis, andpsoriasis. Reversible protein phosphorylation is the main strategy forcontrolling activities of eukaryotic cells. It is estimated that morethan 1000 of the 10,000 proteins active in a typical mammalian cell arephosphorylated. The high energy phosphate, which drives activation, isgenerally transferred from adenosine triphosphate molecules (ATP) to aparticular protein by protein kinases and removed from that protein byprotein phosphatases. Phosphorylation occurs in response toextracellular signals (hormones, neurotransmitters, growth anddifferentiation factors, etc), cell cycle checkpoints, and environmentalor nutritional stresses and is roughly analogous to turning on amolecular switch. When the switch goes on, the appropriate proteinkinase activates a metabolic enzyme, regulatory protein, receptor,cytoskeletal protein, ion channel or pump, or transcription factor.

[0004] The kinases comprise the largest known protein group, asuperfamily of enzymes with widely varied functions and specificities.They are usually named after their substrate, their regulatorymolecules, or some aspect of a mutant phenotype. With regard tosubstrates, the protein kinases may be roughly divided into two groups;those that phosphorylate tyrosine residues (protein tyrosine kinases,PTK) and those that phosphorylate serine or threonine residues(serine/threonine kinases, STK). A few protein kinases have dualspecificity and phosphorylate threonine and tyrosine residues. Almostall kinases contain a similar 250-300 amino acid catalytic domain. TheN-terminal domain, which contains subdomains I-IV, generally folds intoa two-lobed structure, which binds and orients the ATP (or GTP) donormolecule. The larger C terminal lobe, which contains subdomains VI A-XI,binds the protein substrate and carries out the transfer of the gammaphosphate from ATP to the hydroxyl group of a serine, threonine, ortyrosine residue. Subdomain V spans the two lobes.

[0005] The kinases may be categorized into families by the differentamino acid sequences (generally between 5 and 100 residues) located oneither side of, or inserted into loops of, the kinase domain. Theseadded amino acid sequences allow the regulation of each kinase as itrecognizes and interacts with its target protein. The primary structureof the kinase domains is conserved and can be further subdivided into 11subdomains. Each of the 11 subdomains contains specific residues andmotifs or patterns of amino acids that are characteristic of thatsubdomain and are highly conserved (Hardie, G. and Hanks, S. (1995) TheProtein Kinase Facts Books, Vol 1:7-20 Academic Press, San Diego,Calif.).

[0006] The second messenger dependent protein kinases primarily mediatethe effects of second messengers such as cyclic AMP (cAMP), cyclic GMP,inositol triphosphate, phosphatidylinositol, 3,4,5-triphosphate,cyclic-ADPribose, arachidonic acid, diacylglycerol andcalcium-calmodulin. The cyclic-AMP dependent protein kinases (PKA) areimportant members of the STK family. Cyclic-AMP is an intracellularmediator of hormone action in all prokaryotic and animal cells that havebeen studied. Such hormone-induced cellular responses include thyroidhormone secretion, cortisol secretion, progesterone secretion, glycogenbreakdown, bone resorption, and regulation of heart rate and force ofheart muscle contraction. PKA is found in all animal cells and isthought to account for the effects of cyclic-AMP in most of these cells.Altered PKA expression is implicated in a variety of disorders anddiseases including cancer, thyroid disorders, diabetes, atherosclerosis,and cardiovascular disease (Isselbacher, K. J. et al. (1994) Harrison'sPrinciples of Internal Medicine, McGraw-Hill, New York, N.Y., pp.416-431, 1887).

[0007] Calcium-calmodulin (CaM) dependent protein kinases are alsomembers of STK family. Calmodulin is a calcium receptor that mediatesmany calcium regulated processes by binding to target proteins inresponse to the binding of calcium. The principle target protein inthese processes is CaM dependent protein kinases. CaM-kinases areinvolved in regulation of smooth muscle contraction (MLC kinase),glycogen breakdown (phosphorylase kinase), and neurotransmission (CaMkinase I and CaM kinase II). CaM kinase I phosphorylates a variety ofsubstrates including the neurotransmitter related proteins synapsin Iand II, the gene transcription regulator, CREB, and the cystic fibrosisconductance regulator protein, CFTR (Haribabu, B. et al. (1995) EMBOJournal 14:3679-86). CaM II kinase also phosphorylates synapsin atdifferent sites, and controls the synthesis of catecholamines in thebrain through phosphorylation and activation of tyrosine hydroxylase.Many of the CaM kinases are activated by phosphorylation in addition tobinding to CaM. The kinase may autophosphorylate itself, or bephosphorylated by another kinase as part of a “kinase cascade”.

[0008] Another ligand-activated protein kinase is 5′-AMP-activatedprotein kinase (AMPK) (Gao, G. et al. (1996) J. Biol Chem. 15:8675-81).Mammalian AMPK is a regulator of fatty acid and sterol synthesis throughphosphorylation of the enzymes acetyl-CoA carboxylase andhydroxymethylglutaryl-CoA reductase and mediates responses of thesepathways to cellular stresses such as heat shock and depletion ofglucose and ATP. AMPK is a heterotrimeric complex comprised of acatalytic alpha subunit and two non-catalytic beta and gamma subunitsthat are believed to regulate the activity of the alpha subunit.Subunits of AMPK have a much wider distribution in non-lipogenic tissuessuch as brain, heart, spleen, and lung than expected. This distributionsuggests that its role may extend beyond regulation of lipid metabolismalone.

[0009] The mitogen-activated protein kinases (MAP) are also members ofthe STK family. MAP kinases also regulate intracellular signalingpathways. They mediate signal transduction from the cell surface to thenucleus via phosphorylation cascades. Several subgroups have beenidentified, and each manifests different substrate specificities andresponds to distinct extracellular stimuli (Egan, S. E. and Weinberg, R.A. (1993) Nature 365:781-783). MAP kinase signaling pathways are presentin mammalian cells as well as in yeast. The extracellular stimuli thatactivate mammalian pathways include epidermal growth factor (EGF),ultraviolet light, hyperosmolar medium, heat shock, endotoxiclipopolysaccharide (LPS), and pro-inflammatory cytokines such as tumornecrosis factor (TNF) and interleukin-1 (IL-1).

[0010] PRK (proliferation-related kinase) is a serum/cytokine inducibleSTK that is involved in regulation of the cell cycle and cellproliferation in human megakaroytic cells (Li, B. et al. (1996) J. Biol.Chem. 271:19402-8). PRK is related to the polo (derived from humans pologene) family of STKs implicated in cell division. PRK is downregulatedin lung tumor tissue and may be a proto-oncogene whose deregulatedexpression in normal tissue leads to oncogenic transformation. AlteredMAP kinase expression is implicated in a variety of disease conditionsincluding cancer, inflammation, immune disorders, and disordersaffecting growth and development.

[0011] The cyclin-dependent protein kinases (CDKs) are another group ofSTKs that control the progression of cells through the cell cycle.Cyclins are small regulatory proteins that act by binding to andactivating CDKs that then trigger various phases of the cell cycle byphosphorylating and activating selected proteins involved in the mitoticprocess. CDKs are unique in that they require multiple inputs to becomeactivated. In addition to the binding of cyclin, CDK activation requiresthe phosphorylation of a specific threonine residue and thedephosphorylation of a specific tyrosine residue.

[0012] Protein tyrosine kinases, PTKs, specifically phosphorylatetyrosine residues on their target proteins and may be divided intotransmembrane, receptor PTKs and nontransmembrane, non-receptor PTKs.Transmembrane protein-tyrosine kinases are receptors for most growthfactors. Binding of growth factor to the receptor activates the transferof a phosphate group from ATP to selected tyrosine side chains of thereceptor and other specific proteins. Growth factors (GF) associatedwith receptor PTKs include; epidermal GF, platelet-derived GF,fibroblast GF, hepatocyte GF, insulin and insulin-like GFs, nerve GF,vascular endothelial GF, and macrophage colony stimulating factor.

[0013] Non-receptor PTKs lack transmembrane regions and, instead, formcomplexes with the intracellular regions of cell surface receptors. Suchreceptors that function through non-receptor PTKs include those forcytokines, hormones (growth hormone and prolactin) and antigen-specificreceptors on T and B lymphocytes.

[0014] Many of these PTKs were first identified as the products ofmutant oncogenes in cancer cells where their activation was no longersubject to normal cellular controls. In fact, about one third of theknown oncogenes encode PTKs, and it is well known that cellulartransformation (oncogenesis) is often accompanied by increased tyrosinephosphorylation activity (Carbonneau H and Tonks NK (1992) Annu. Rev.Cell. Biol. 8:463-93). Regulation of PTK activity may therefore be animportant strategy in controlling some types of cancer.

[0015] Serum/Glucocorticoid Regulated Kinase (SGK)

[0016] The novel human protein, and encoding gene, provided by thepresent invention is a novel serine/threonine protein kinase that sharesthe highest degree of sequence similarity with serum/glucocorticoidregulated kinase (SGK; sgk) (see the alignment of SEQ ID NO: 2 andGenbank gi:14756346 provided in FIG. 2). Furthermore, like the gene ofthe present invention, the SGK gene also maps to human chromosome 6(Waldegger et al., Genomics 51: 299-302, 1998).

[0017] Human SGK shares 98% sequence identity with rat sgk, expressionof which in mammary tumor cells is stimulated by glucocorticoid hormonesand serum (Webster et al., Molec. Cell. Biol. 13: 2031-2040, 1993).Transcription of SGK is rapidly raised upon exposure to hypertonicconditions and decreased upon exposure to hypotonic conditions(furthermore, cell volume rather than osmolarity was shown to beresponsible for regulation of SGK expression), suggesting that SGK mayprovide a functional link between the cellular hydration state andmetabolic control (Waldegger et al., Proc. Nat. Acad. Sci. 94:4440-4445, 1997). Changes in hepatocyte cell volume (e.g., in responseto anisotonicity, concentrative substrate uptake, oxidative stress, andhormonal influence) are known to influence hepatocellular metabolism andgene expression (Waldegger et al., Proc. Nat. Acad. Sci. 94: 4440-4445,1997).

[0018] Furthermore, transforming growth factor-beta (TGFB1) stimulatesSGK expression, and TGFB1 is involved in the pathophysiology of diabeticcomplications such as diabetic nephopathy. SGK expression issignificantly increased by excessive extracellular glucoseconcentrations in diabetic nephropathy, and is particularly highlyexpressed in mesangial cells, interstitial cells, and cells of the loopof Henle and distal tubules. This increased SGK expression stimulatesrenal tubular sodium transport (Lang et al., Proc. Nat. Acad. Sci. 97:8157-8162, 2000).

[0019] Kinase proteins, particularly members of the serine/threonineprotein kinase subfamily, are a major target for drug action anddevelopment. Accordingly, it is valuable to the field of pharmaceuticaldevelopment to identify and characterize previously unknown members ofthis subfamily of kinase proteins. The present invention advances thestate of the art by providing previously unidentified human kinaseproteins that have homology to members of the serine/threonine proteinkinase subfamily.

SUMMARY OF THE INVENTION

[0020] The present invention is based in part on the identification ofamino acid sequences of human kinase peptides and proteins that arerelated to the serine/threonine protein kinase subfamily, as well asallelic variants and other mammalian orthologs thereof. These uniquepeptide sequences, and nucleic acid sequences that encode thesepeptides, can be used as models for the development of human therapeutictargets, aid in the identification of therapeutic proteins, and serve astargets for the development of human therapeutic agents that modulatekinase activity in cells and tissues that express the kinase.Experimental data as provided in FIG. 1 indicates expression in thebrain (hypothalamus and hippocampus), placenta, and mammary gland.

DESCRIPTION OF THE FIGURE SHEETS

[0021]FIG. 1 provides the nucleotide sequence of a transcript sequencethat encodes the kinase protein of the present invention. (SEQ ID NO: 1)In addition, structure and functional information is provided, such asATG start, stop and tissue distribution, where available, that allowsone to readily determine specific uses of inventions based on thismolecular sequence. Experimental data as provided in FIG. 1 indicatesexpression in the brain (hypothalamus and hippocampus), placenta, andmammary gland.

[0022]FIG. 2 provides the predicted amino acid sequence of the kinase ofthe present invention. (SEQ ID NO: 2) In addition structure andfunctional information such as protein family, function, andmodification sites is provided where available, allowing one to readilydetermine specific uses of inventions based on this molecular sequence.

[0023]FIG. 3 provides genomic sequences that span the gene encoding thekinase protein of the present invention. (SEQ ID NO: 3) In additionstructure and functional information, such as intron/exon structure,promoter location, etc., is provided where available, allowing one toreadily determine specific uses of inventions based on this molecularsequence. As illustrated in FIG. 3, one SNP was identified (A738G).

DETAILED DESCRIPTION OF THE INVENTION

[0024] General Description

[0025] The present invention is based on the sequencing of the humangenome. During the sequencing and assembly of the human genome, analysisof the sequence information revealed previously unidentified fragmentsof the human genome that encode peptides that share structural and/orsequence homology to protein/peptide/domains identified andcharacterized within the art as being a kinase protein or part of akinase protein and are related to the serine/threonine protein kinasesubfamily. Utilizing these sequences, additional genomic sequences wereassembled and transcript and/or cDNA sequences were isolated andcharacterized. Based on this analysis, the present invention providesamino acid sequences of human kinase peptides and proteins that arerelated to the serine/threonine protein kinase subfamily, nucleic acidsequences in the form of transcript sequences, cDNA sequences and/orgenomic sequences that encode these kinase peptides and proteins,nucleic acid variation (allelic information), tissue distribution ofexpression, and information about the closest art knownprotein/peptide/domain that has structural or sequence homology to thekinase of the present invention.

[0026] In addition to being previously unknown, the peptides that areprovided in the present invention are selected based on their ability tobe used for the development of commercially important products andservices. Specifically, the present peptides are selected based onhomology and/or structural relatedness to known kinase proteins of theserine/threonine protein kinase subfamily and the expression patternobserved. Experimental data as provided in FIG. 1 indicates expressionin the brain (hypothalamus and hippocampus), placenta, and mammarygland. The art has clearly established the commercial importance ofmembers of this family of proteins and proteins that have expressionpatterns similar to that of the present gene. Some of the more specificfeatures of the peptides of the present invention, and the uses thereof,are described herein, particularly in the Background of the Inventionand in the annotation provided in the Figures, and/or are known withinthe art for each of the known serine/threonine protein kinase family orsubfamily of kinase proteins.

[0027] Specific Embodiments

[0028] Peptide Molecules

[0029] The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being members of thekinase family of proteins and are related to the serine/threonineprotein kinase subfamily (protein sequences are provided in FIG. 2,transcript/cDNA sequences are provided in FIG. 1 and genomic sequencesare provided in FIG. 3). The peptide sequences provided in FIG. 2, aswell as the obvious variants described herein, particularly allelicvariants as identified herein and using the information in FIG. 3, willbe referred herein as the kinase peptides of the present invention,kinase peptides, or peptides/proteins of the present invention.

[0030] The present invention provides isolated peptide and proteinmolecules that consist of, consist essentially of, or comprise the aminoacid sequences of the kinase peptides disclosed in the FIG. 2, (encodedby the nucleic acid molecule shown in FIG. 1, transcript/cDNA or FIG. 3,genomic sequence), as well as all obvious variants of these peptidesthat are within the art to make and use. Some of these variants aredescribed in detail below.

[0031] As used herein, a peptide is said to be “isolated” or “purified”when it is substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

[0032] In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

[0033] The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of thekinase peptide having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

[0034] The isolated kinase peptide can be purified from cells thatnaturally express it, purified from cells that have been altered toexpress it (recombinant), or synthesized using known protein synthesismethods. Experimental data as provided in FIG. 1 indicates expression inthe brain (hypothalamus and hippocampus), placenta, and mammary gland.For example, a nucleic acid molecule encoding the kinase peptide iscloned into an expression vector, the expression vector introduced intoa host cell and the protein expressed in the host cell. The protein canthen be isolated from the cells by an appropriate purification schemeusing standard protein purification techniques. Many of these techniquesare described in detail below.

[0035] Accordingly, the present invention provides proteins that consistof the amino acid sequences provided in FIG. 2 (SEQ ID NO: 2), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NO: 1) and the genomic sequences provided inFIG. 3 (SEQ ID NO: 3). The amino acid sequence of such a protein isprovided in FIG. 2. A protein consists of an amino acid sequence whenthe amino acid sequence is the final amino acid sequence of the protein.

[0036] The present invention further provides proteins that consistessentially of the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acidsequences shown in FIG. 1 (SEQ ID NO: 1) and the genomic sequencesprovided in FIG. 3 (SEQ ID NO: 3). A protein consists essentially of anamino acid sequence when such an amino acid sequence is present withonly a few additional amino acid residues, for example from about 1 toabout 100 or so additional residues, typically from 1 to about 20additional residues in the final protein.

[0037] The present invention further provides proteins that comprise theamino acid sequences provided in FIG. 2 (SEQ ID NO: 2), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NO: 1) and the genomic sequences provided in FIG. 3 (SEQID NO: 3). A protein comprises an amino acid sequence when the aminoacid sequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein can be only the peptide or haveadditional amino acid molecules, such as amino acid residues (contiguousencoded sequence) that are naturally associated with it or heterologousamino acid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the kinase peptides of the present invention are thenaturally occurring mature proteins. A brief description of how varioustypes of these proteins can be made/isolated is provided below.

[0038] The kinase peptides of the present invention can be attached toheterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a kinase peptide operativelylinked to a heterologous protein having an amino acid sequence notsubstantially homologous to the kinase peptide. “Operatively linked”indicates that the kinase peptide and the heterologous protein are fusedin-frame. The heterologous protein can be fused to the N-terminus orC-terminus of the kinase peptide.

[0039] In some uses, the fusion protein does not affect the activity ofthe kinase peptide per se. For example, the fusion protein can include,but is not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins,particularly poly-His fusions, can facilitate the purification ofrecombinant kinase peptide. In certain host cells (e.g., mammalian hostcells), expression and/or secretion of a protein can be increased byusing a heterologous signal sequence.

[0040] A chimeric or fusion protein can be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent protein sequences are ligated together in-frame in accordancewith conventional techniques. In another embodiment, the fusion gene canbe synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (seeAusubel et al., Current Protocols in Molecular Biology, 1992). Moreover,many expression vectors are commercially available that already encode afusion moiety (e.g., a GST protein). A kinase peptide-encoding nucleicacid can be cloned into such an expression vector such that the fusionmoiety is linked in-frame to the kinase peptide.

[0041] As mentioned above, the present invention also provides andenables obvious variants of the amino acid sequence of the proteins ofthe present invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

[0042] Such variants can readily be identified/made using moleculartechniques and the sequence information disclosed herein. Further, suchvariants can readily be distinguished from other peptides based onsequence and/or structural homology to the kinase peptides of thepresent invention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

[0043] To determine the percent identity of two amino acid sequences ortwo nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%,80%, or 90% or more of the length of a reference sequence is aligned forcomparison purposes. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

[0044] The comparison of sequences and determination of percent identityand similarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al.,Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Myers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

[0045] The nucleic acid and protein sequences of the present inventioncan further be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

[0046] Full-length pre-processed forms, as well as mature processedforms, of proteins that comprise one of the peptides of the presentinvention can readily be identified as having complete sequence identityto one of the kinase peptides of the present invention as well as beingencoded by the same genetic locus as the kinase peptide provided herein.As indicated by the data presented in FIG. 3, the chromosome mapposition was determined to be on human chromosome 6.

[0047] Allelic variants of a kinase peptide can readily be identified asbeing a human protein having a high degree (significant) of sequencehomology/identity to at least a portion of the kinase peptide as well asbeing encoded by the same genetic locus as the kinase peptide providedherein. Genetic locus can readily be determined based on the genomicinformation provided in FIG. 3, such as the genomic sequence mapped tothe reference human. As indicated by the data presented in FIG. 3, thechromosome map position was determined to be on human chromosome 6. Asused herein, two proteins (or a region of the proteins) have significanthomology when the amino acid sequences are typically at least about70-80%, 80-90%, and more typically at least about 90-95% or morehomologous. A significantly homologous amino acid sequence, according tothe present invention, will be encoded by a nucleic acid sequence thatwill hybridize to a kinase peptide encoding nucleic acid molecule understringent conditions as more fully described below.

[0048]FIG. 3 provides information on a polymorphism (A738G) that wasidentified in the gene encoding the kinase proteins of the presentinvention.

[0049] Paralogs of a kinase peptide can readily be identified as havingsome degree of significant sequence homology/identity to at least aportion of the kinase peptide, as being encoded by a gene from humans,and as having similar activity or function. Two proteins will typicallybe considered paralogs when the amino acid sequences are typically atleast about 60% or greater, and more typically at least about 70% orgreater homology through a given region or domain. Such paralogs will beencoded by a nucleic acid sequence that will hybridize to a kinasepeptide encoding nucleic acid molecule under moderate to stringentconditions as more fully described below.

[0050] Orthologs of a kinase peptide can readily be identified as havingsome degree of significant sequence homology/identity to at least aportion of the kinase peptide as well as being encoded by a gene fromanother organism. Preferred orthologs will be isolated from mammals,preferably primates, for the development of human therapeutic targetsand agents. Such orthologs will be encoded by a nucleic acid sequencethat will hybridize to a kinase peptide encoding nucleic acid moleculeunder moderate to stringent conditions, as more fully described below,depending on the degree of relatedness of the two organisms yielding theproteins.

[0051] Non-naturally occurring variants of the kinase peptides of thepresent invention can readily be generated using recombinant techniques.Such variants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the kinase peptide. Forexample, one class of substitutions are conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in a kinase peptide by another amino acid of like characteristics.Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu, and Ile;interchange of the hydroxyl residues Ser and Thr; exchange of the acidicresidues Asp and Glu; substitution between the amide residues Asn andGln; exchange of the basic residues Lys and Arg; and replacements amongthe aromatic residues Phe and Tyr. Guidance concerning which amino acidchanges are likely to be phenotypically silent are found in Bowie etal., Science 247:1306-1310 (1990).

[0052] Variant kinase peptides can be fully functional or can lackfunction in one or more activities, e.g. ability to bind substrate,ability to phosphorylate substrate, ability to mediate signaling, etc.Fully functional variants typically contain only conservative variationor variation in non-critical residues or in non-critical regions. FIG. 2provides the result of protein analysis and can be used to identifycritical domains/regions. Functional variants can also containsubstitution of similar amino acids that result in no change or aninsignificant change in function. Alternatively, such substitutions maypositively or negatively affect function to some degree.

[0053] Non-functional variants typically contain one or morenon-conservative amino acid substitutions, deletions, insertions,inversions, or truncation or a substitution, insertion, inversion, ordeletion in a critical residue or critical region.

[0054] Amino acids that are essential for function can be identified bymethods known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085(1989)), particularly using the results provided in FIG. 2. The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as kinase activity or in assays such as an in vitroproliferative activity. Sites that are critical for bindingpartner/substrate binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al.Science 255:306-312 (1992)).

[0055] The present invention further provides fragments of the kinasepeptides, in addition to proteins and peptides that comprise and consistof such fragments, particularly those comprising the residues identifiedin FIG. 2. The fragments to which the invention pertains, however, arenot to be construed as encompassing fragments that may be disclosedpublicly prior to the present invention.

[0056] As used herein, a fragment comprises at least 8, 10, 12, 14, 16,or more contiguous amino acid residues from a kinase peptide. Suchfragments can be chosen based on the ability to retain one or more ofthe biological activities of the kinase peptide or could be chosen forthe ability to perform a function, e.g. bind a substrate or act as animmunogen. Particularly important fragments are biologically activefragments, peptides that are, for example, about 8 or more amino acidsin length. Such fragments will typically comprise a domain or motif ofthe kinase peptide, e.g., active site, a transmembrane domain or asubstrate-binding domain. Further, possible fragments include, but arenot limited to, domain or motif containing fragments, soluble peptidefragments, and fragments containing immunogenic structures. Predicteddomains and functional sites are readily identifiable by computerprograms well known and readily available to those of skill in the art(e.g., PROSITE analysis). The results of one such analysis are providedin FIG. 2.

[0057] Polypeptides often contain amino acids other than the 20 aminoacids commonly referred to as the 20 naturally occurring amino acids.Further, many amino acids, including the terminal amino acids, may bemodified by natural processes, such as processing and otherpost-translational modifications, or by chemical modification techniqueswell known in the art. Common modifications that occur naturally inkinase peptides are described in basic texts, detailed monographs, andthe research literature, and they are well known to those of skill inthe art (some of these features are identified in FIG. 2).

[0058] Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

[0059] Such modifications are well known to those of skill in the artand have been described in great detail in the scientific literature.Several particularly common modifications, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, for instance, are described in mostbasic texts, such as Proteins—Structure and Molecular Properties, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. NY. Acad. Sci. 663:48-62(1992)).

[0060] Accordingly, the kinase peptides of the present invention alsoencompass derivatives or analogs in which a substituted amino acidresidue is not one encoded by the genetic code, in which a substituentgroup is included, in which the mature kinase peptide is fused withanother compound, such as a compound to increase the half-life of thekinase peptide (for example, polyethylene glycol), or in which theadditional amino acids are fused to the mature kinase peptide, such as aleader or secretory sequence or a sequence for purification of themature kinase peptide or a pro-protein sequence.

[0061] Protein/Peptide Uses

[0062] The proteins of the present invention can be used in substantialand specific assays related to the functional information provided inthe Figures; to raise antibodies or to elicit another immune response;as a reagent (including the labeled reagent) in assays designed toquantitatively determine levels of the protein (or its binding partneror ligand) in biological fluids; and as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state). Where the protein binds or potentially binds toanother protein or ligand (such as, for example, in a kinase-effectorprotein interaction or kinase-ligand interaction), the protein can beused to identify the binding partner/ligand so as to develop a system toidentify inhibitors of the binding interaction. Any or all of these usesare capable of being developed into reagent grade or kit format forcommercialization as commercial products.

[0063] Methods for performing the uses listed above are well known tothose skilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

[0064] The potential uses of the peptides of the present invention arebased primarily on the source of the protein as well as the class/actionof the protein. For example, kinases isolated from humans and theirhuman/mammalian orthologs serve as targets for identifying agents foruse in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the kinase. Experimental data as providedin FIG. 1 indicates that kinase proteins of the present invention areexpressed in the brain (hypothalamus and hippocampus), placenta, andmammary gland, as indicated by virtual northern blot analysis. A largepercentage of pharmaceutical agents are being developed that modulatethe activity of kinase proteins, particularly members of theserine/threonine protein kinase subfamily (see Background of theInvention). The structural and functional information provided in theBackground and Figures provide specific and substantial uses for themolecules of the present invention, particularly in combination with theexpression information provided in FIG. 1. Experimental data as providedin FIG. 1 indicates expression in the brain (hypothalamus andhippocampus), placenta, and mammary gland. Such uses can readily bedetermined using the information provided herein, that which is known inthe art, and routine experimentation.

[0065] The proteins of the present invention (including variants andfragments that may have been disclosed prior to the present invention)are useful for biological assays related to kinases that are related tomembers of the serine/threonine protein kinase subfamily. Such assaysinvolve any of the known kinase functions or activities or propertiesuseful for diagnosis and treatment of kinase-related conditions that arespecific for the subfamily of kinases that the one of the presentinvention belongs to, particularly in cells and tissues that express thekinase. Experimental data as provided in FIG. 1 indicates that kinaseproteins of the present invention are expressed in the brain(hypothalamus and hippocampus), placenta, and mammary gland, asindicated by virtual northern blot analysis.

[0066] The proteins of the present invention are also useful in drugscreening assays, in cell-based or cell-free systems. Cell-based systemscan be native, i.e., cells that normally express the kinase, as a biopsyor expanded in cell culture. Experimental data as provided in FIG. 1indicates expression in the brain (hypothalamus and hippocampus),placenta, and mammary gland. In an alternate embodiment, cell-basedassays involve recombinant host cells expressing the kinase protein.

[0067] The polypeptides can be used to identify compounds that modulatekinase activity of the protein in its natural state or an altered formthat causes a specific disease or pathology associated with the kinase.Both the kinases of the present invention and appropriate variants andfragments can be used in high-throughput screens to assay candidatecompounds for the ability to bind to the kinase. These compounds can befurther screened against a functional kinase to determine the effect ofthe compound on the kinase activity. Further, these compounds can betested in animal or invertebrate systems to determineactivity/effectiveness. Compounds can be identified that activate(agonist) or inactivate (antagonist) the kinase to a desired degree.

[0068] Further, the proteins of the present invention can be used toscreen a compound for the ability to stimulate or inhibit interactionbetween the kinase protein and a molecule that normally interacts withthe kinase protein, e.g. a substrate or a component of the signalpathway that the kinase protein normally interacts (for example, anotherkinase). Such assays typically include the steps of combining the kinaseprotein with a candidate compound under conditions that allow the kinaseprotein, or fragment, to interact with the target molecule, and todetect the formation of a complex between the protein and the target orto detect the biochemical consequence of the interaction with the kinaseprotein and the target, such as any of the associated effects of signaltransduction such as protein phosphorylation, cAMP turnover, andadenylate cyclase activation, etc.

[0069] Candidate compounds include, for example, 1) peptides such assoluble peptides, including Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g., Lam et al., Nature 354:82-84(1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

[0070] One candidate compound is a soluble fragment of the receptor thatcompetes for substrate binding. Other candidate compounds include mutantkinases or appropriate fragments containing mutations that affect kinasefunction and thus compete for substrate. Accordingly, a fragment thatcompetes for substrate, for example with a higher affinity, or afragment that binds substrate but does not allow release, is encompassedby the invention.

[0071] The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) kinase activity. Theassays typically involve an assay of events in the signal transductionpathway that indicate kinase activity. Thus, the phosphorylation of asubstrate, activation of a protein, a change in the expression of genesthat are up- or down-regulated in response to the kinase proteindependent signal cascade can be assayed.

[0072] Any of the biological or biochemical functions mediated by thekinase can be used as an endpoint assay. These include all of thebiochemical or biochemical/biological events described herein, in thereferences cited herein, incorporated by reference for these endpointassay targets, and other functions known to those of ordinary skill inthe art or that can be readily identified using the information providedin the Figures, particularly FIG. 2. Specifically, a biological functionof a cell or tissues that expresses the kinase can be assayed.Experimental data as provided in FIG. 1 indicates that kinase proteinsof the present invention are expressed in the brain (hypothalamus andhippocampus), placenta, and mammary gland, as indicated by virtualnorthern blot analysis.

[0073] Binding and/or activating compounds can also be screened by usingchimeric kinase proteins in which the amino terminal extracellulardomain, or parts thereof, the entire transmembrane domain or subregions,such as any of the seven transmembrane segments or any of theintracellular or extracellular loops and the carboxy terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a substrate-binding region can beused that interacts with a different substrate then that which isrecognized by the native kinase. Accordingly, a different set of signaltransduction components is available as an end-point assay foractivation. This allows for assays to be performed in other than thespecific host cell from which the kinase is derived.

[0074] The proteins of the present invention are also useful incompetition binding assays in methods designed to discover compoundsthat interact with the kinase (e.g. binding partners and/or ligands).Thus, a compound is exposed to a kinase polypeptide under conditionsthat allow the compound to bind or to otherwise interact with thepolypeptide. Soluble kinase polypeptide is also added to the mixture. Ifthe test compound interacts with the soluble kinase polypeptide, itdecreases the amount of complex formed or activity from the kinasetarget. This type of assay is particularly useful in cases in whichcompounds are sought that interact with specific regions of the kinase.Thus, the soluble polypeptide that competes with the target kinaseregion is designed to contain peptide sequences corresponding to theregion of interest.

[0075] To perform cell free drug screening assays, it is sometimesdesirable to immobilize either the kinase protein, or fragment, or itstarget molecule to facilitate separation of complexes from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay.

[0076] Techniques for immobilizing proteins on matrices can be used inthe drug screening assays. In one embodiment, a fusion protein can beprovided which adds a domain that allows the protein to be bound to amatrix. For example, glutathione-S-transferase fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the cell lysates (e.g., ³⁵S-labeled) and the candidatecompound, and the mixture incubated under conditions conducive tocomplex formation (e.g., at physiological conditions for salt and pH).Following incubation, the beads are washed to remove any unbound label,and the matrix immobilized and radiolabel determined directly, or in thesupernatant after the complexes are dissociated. Alternatively, thecomplexes can be dissociated from the matrix, separated by SDS-PAGE, andthe level of kinase-binding protein found in the bead fractionquantitated from the gel using standard electrophoretic techniques. Forexample, either the polypeptide or its target molecule can beimmobilized utilizing conjugation of biotin and streptavidin usingtechniques well known in the art. Alternatively, antibodies reactivewith the protein but which do not interfere with binding of the proteinto its target molecule can be derivatized to the wells of the plate, andthe protein trapped in the wells by antibody conjugation. Preparationsof a kinase-binding protein and a candidate compound are incubated inthe kinase protein-presenting wells and the amount of complex trapped inthe well can be quantitated. Methods for detecting such complexes, inaddition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with thekinase protein target molecule, or which are reactive with kinaseprotein and compete with the target molecule, as well as enzyme-linkedassays which rely on detecting an enzymatic activity associated with thetarget molecule.

[0077] Agents that modulate one of the kinases of the present inventioncan be identified using one or more of the above assays, alone or incombination. It is generally preferable to use a cell-based or cell freesystem first and then confirm activity in an animal or other modelsystem. Such model systems are well known in the art and can readily beemployed in this context.

[0078] Modulators of kinase protein activity identified according tothese drug screening assays can be used to treat a subject with adisorder mediated by the kinase pathway, by treating cells or tissuesthat express the kinase. Experimental data as provided in FIG. 1indicates expression in the brain (hypothalamus and hippocampus),placenta, and mammary gland. These methods of treatment include thesteps of administering a modulator of kinase activity in apharmaceutical composition to a subject in need of such treatment, themodulator being identified as described herein.

[0079] In yet another aspect of the invention, the kinase proteins canbe used as “bait proteins” in a two-hybrid assay or three-hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with the kinase and are involved in kinase activity.Such kinase-binding proteins are also likely to be involved in thepropagation of signals by the kinase proteins or kinase targets as, forexample, downstream elements of a kinase-mediated signaling pathway.Alternatively, such kinase-binding proteins are likely to be kinaseinhibitors.

[0080] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a kinase proteinis fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming akinase-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the kinase protein.

[0081] This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a kinase-modulating agent, an antisense kinasenucleic acid molecule, a kinase-specific antibody, or a kinase-bindingpartner) can be used in an animal or other model to determine theefficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal or other model to determine the mechanism of action of such anagent. Furthermore, this invention pertains to uses of novel agentsidentified by the above-described screening assays for treatments asdescribed herein.

[0082] The kinase proteins of the present invention are also useful toprovide a target for diagnosing a disease or predisposition to diseasemediated by the peptide. Accordingly, the invention provides methods fordetecting the presence, or levels of, the protein (or encoding mRNA) ina cell, tissue, or organism. Experimental data as provided in FIG. 1indicates expression in the brain (hypothalamus and hippocampus),placenta, and mammary gland. The method involves contacting a biologicalsample with a compound capable of interacting with the kinase proteinsuch that the interaction can be detected. Such an assay can be providedin a single detection format or a multi-detection format such as anantibody chip array.

[0083] One agent for detecting a protein in a sample is an antibodycapable of selectively binding to protein. A biological sample includestissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject.

[0084] The peptides of the present invention also provide targets fordiagnosing active protein activity, disease, or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered kinase activity in cell-based orcell-free assay, alteration in substrate or antibody-binding pattern,altered isoelectric point, direct amino acid sequencing, and any otherof the known assay techniques useful for detecting mutations in aprotein. Such an assay can be provided in a single detection format or amulti-detection format such as an antibody chip array.

[0085] In vitro techniques for detection of peptide include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence using a detection reagent,such as an antibody or protein binding agent. Alternatively, the peptidecan be detected in vivo in a subject by introducing into the subject alabeled anti-peptide antibody or other types of detection agent. Forexample, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques. Particularly useful are methods that detect the allelicvariant of a peptide expressed in a subject and methods which detectfragments of a peptide in a sample.

[0086] The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the kinase protein in which one ormore of the kinase functions in one population is different from thosein another population. The peptides thus allow a target to ascertain agenetic predisposition that can affect treatment modality. Thus, in aligand-based treatment, polymorphism may give rise to amino terminalextracellular domains and/or other substrate-binding regions that aremore or less active in substrate binding, and kinase activation.Accordingly, substrate dosage would necessarily be modified to maximizethe therapeutic effect within a given population containing apolymorphism. As an alternative to genotyping, specific polymorphicpeptides could be identified.

[0087] The peptides are also useful for treating a disordercharacterized by an absence of, inappropriate, or unwanted expression ofthe protein. Experimental data as provided in FIG. 1 indicatesexpression in the brain (hypothalamus and hippocampus), placenta, andmammary gland. Accordingly, methods for treatment include the use of thekinase protein or fragments.

[0088] Antibodies

[0089] The invention also provides antibodies that selectively bind toone of the peptides of the present invention, a protein comprising sucha peptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

[0090] As used herein, an antibody is defined in terms consistent withthat recognized within the art: they are multi-subunit proteins producedby a mammalian organism in response to an antigen challenge. Theantibodies of the present invention include polyclonal antibodies andmonoclonal antibodies, as well as fragments of such antibodies,including, but not limited to, Fab or F(ab′)₂, and Fv fragments.

[0091] Many methods are known for generating and/or identifyingantibodies to a given target peptide. Several such methods are describedby Harlow, Antibodies, Cold Spring Harbor Press, (1989).

[0092] In general, to generate antibodies, an isolated peptide is usedas an immunogen and is administered to a mammalian organism, such as arat, rabbit or mouse. The full-length protein, an antigenic peptidefragment or a fusion protein can be used. Particularly importantfragments are those covering functional domains, such as the domainsidentified in FIG. 2, and domain of sequence homology or divergenceamongst the family, such as those that can readily be identified usingprotein alignment methods and as presented in the Figures.

[0093] Antibodies are preferably prepared from regions or discretefragments of the kinase proteins. Antibodies can be prepared from anyregion of the peptide as described herein. However, preferred regionswill include those involved in function/activity and/or kinase/bindingpartner interaction. FIG. 2 can be used to identify particularlyimportant regions while sequence alignment can be used to identifyconserved and unique sequence fragments.

[0094] An antigenic fragment will typically comprise at least 8contiguous amino acid residues. The antigenic peptide can comprise,however, at least 10, 12, 14, 16 or more amino acid residues. Suchfragments can be selected on a physical property, such as fragmentscorrespond to regions that are located on the surface of the protein,e.g., hydrophilic regions or can be selected based on sequenceuniqueness (see FIG. 2).

[0095] Detection on an antibody of the present invention can befacilitated by coupling (i.e., physically linking) the antibody to adetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

[0096] Antibody Uses

[0097] The antibodies can be used to isolate one of the proteins of thepresent invention by standard techniques, such as affinitychromatography or immunoprecipitation. The antibodies can facilitate thepurification of the natural protein from cells and recombinantlyproduced protein expressed in host cells. In addition, such antibodiesare useful to detect the presence of one of the proteins of the presentinvention in cells or tissues to determine the pattern of expression ofthe protein among various tissues in an organism and over the course ofnormal development. Experimental data as provided in FIG. 1 indicatesthat kinase proteins of the present invention are expressed in the brain(hypothalamus and hippocampus), placenta, and mammary gland, asindicated by virtual northern blot analysis. Further, such antibodiescan be used to detect protein in situ, in vitro, or in a cell lysate orsupernatant in order to evaluate the abundance and pattern ofexpression. Also, such antibodies can be used to assess abnormal tissuedistribution or abnormal expression during development or progression ofa biological condition. Antibody detection of circulating fragments ofthe full length protein can be used to identify turnover.

[0098] Further, the antibodies can be used to assess expression indisease states such as in active stages of the disease or in anindividual with a predisposition toward disease related to the protein'sfunction. When a disorder is caused by an inappropriate tissuedistribution, developmental expression, level of expression of theprotein, or expressed/processed form, the antibody can be preparedagainst the normal protein. Experimental data as provided in FIG. 1indicates expression in the brain (hypothalamus and hippocampus),placenta, and mammary gland. If a disorder is characterized by aspecific mutation in the protein, antibodies specific for this mutantprotein can be used to assay for the presence of the specific mutantprotein.

[0099] The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data as provided in FIG. 1 indicates expression in thebrain (hypothalamus and hippocampus), placenta, and mammary gland. Thediagnostic uses can be applied, not only in genetic testing, but also inmonitoring a treatment modality. Accordingly, where treatment isultimately aimed at correcting expression level or the presence ofaberrant sequence and aberrant tissue distribution or developmentalexpression, antibodies directed against the protein or relevantfragments can be used to monitor therapeutic efficacy.

[0100] Additionally, antibodies are useful in pharmacogenomic analysis.Thus, antibodies prepared against polymorphic proteins can be used toidentify individuals that require modified treatment modalities. Theantibodies are also useful as diagnostic tools as an immunologicalmarker for aberrant protein analyzed by electrophoretic mobility,isoelectric point, tryptic peptide digest, and other physical assaysknown to those in the art.

[0101] The antibodies are also useful for tissue typing. Experimentaldata as provided in FIG. 1 indicates expression in the brain(hypothalamus and hippocampus), placenta, and mammary gland. Thus, wherea specific protein has been correlated with expression in a specifictissue, antibodies that are specific for this protein can be used toidentify a tissue type.

[0102] The antibodies are also useful for inhibiting protein function,for example, blocking the binding of the kinase peptide to a bindingpartner such as a substrate. These uses can also be applied in atherapeutic context in which treatment involves inhibiting the protein'sfunction. An antibody can be used, for example, to block binding, thusmodulating (agonizing or antagonizing) the peptides activity. Antibodiescan be prepared against specific fragments containing sites required forfunction or against intact protein that is associated with a cell orcell membrane. See FIG. 2 for structural information relating to theproteins of the present invention.

[0103] The invention also encompasses kits for using antibodies todetect the presence of a protein in a biological sample. The kit cancomprise antibodies such as a labeled or labelable antibody and acompound or agent for detecting protein in a biological sample; meansfor determining the amount of protein in the sample; means for comparingthe amount of protein in the sample with a standard; and instructionsfor use. Such a kit can be supplied to detect a single protein orepitope or can be configured to detect one of a multitude of epitopes,such as in an antibody detection array. Arrays are described in detailbelow for nuleic acid arrays and similar methods have been developed forantibody arrays.

[0104] Nucleic Acid Molecules

[0105] The present invention further provides isolated nucleic acidmolecules that encode a kinase peptide or protein of the presentinvention (cDNA, transcript and genomic sequence). Such nucleic acidmolecules will consist of, consist essentially of, or comprise anucleotide sequence that encodes one of the kinase peptides of thepresent invention, an allelic variant thereof, or an ortholog or paralogthereof.

[0106] As used herein, an “isolated” nucleic acid molecule is one thatis separated from other nucleic acid present in the natural source ofthe nucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. However, there canbe some flanking nucleotide sequences, for example up to about 5 KB, 4KB, 3 KB, 2 KB, or 1 KB or less, particularly contiguous peptideencoding sequences and peptide encoding sequences within the same genebut separated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

[0107] Moreover, an “isolated” nucleic acid molecule, such as atranscript/cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orchemical precursors or other chemicals when chemically synthesized.However, the nucleic acid molecule can be fused to other coding orregulatory sequences and still be considered isolated.

[0108] For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified partially or substantially) DNA molecules in solution. IsolatedRNA molecules include in vivo or in vitro RNA transcripts of theisolated DNA molecules of the present invention. Isolated nucleic acidmolecules according to the present invention further include suchmolecules produced synthetically.

[0109] Accordingly, the present invention provides nucleic acidmolecules that consist of the nucleotide sequence shown in FIG. 1 or 3(SEQ ID NO: 1, transcript sequence and SEQ ID NO: 3, genomic sequence),or any nucleic acid molecule that encodes the protein provided in FIG.2, SEQ ID NO: 2. A nucleic acid molecule consists of a nucleotidesequence when the nucleotide sequence is the complete nucleotidesequence of the nucleic acid molecule.

[0110] The present invention further provides nucleic acid moleculesthat consist essentially of the nucleotide sequence shown in FIG. 1 or 3(SEQ ID NO: 1, transcript sequence and SEQ ID NO: 3, genomic sequence),or any nucleic acid molecule that encodes the protein provided in FIG.2, SEQ ID NO: 2. A nucleic acid molecule consists essentially of anucleotide sequence when such a nucleotide sequence is present with onlya few additional nucleic acid residues in the final nucleic acidmolecule.

[0111] The present invention further provides nucleic acid moleculesthat comprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO: 3, genomic sequence), or anynucleic acid molecule that encodes the protein provided in FIG. 2, SEQID NO: 2. A nucleic acid molecule comprises a nucleotide sequence whenthe nucleotide sequence is at least part of the final nucleotidesequence of the nucleic acid molecule. In such a fashion, the nucleicacid molecule can be only the nucleotide sequence or have additionalnucleic acid residues, such as nucleic acid residues that are naturallyassociated with it or heterologous nucleotide sequences. Such a nucleicacid molecule can have a few additional nucleotides or can comprisesseveral hundred or more additional nucleotides. A brief description ofhow various types of these nucleic acid molecules can be readilymade/isolated is provided below.

[0112] In FIGS. 1 and 3, both coding and non-coding sequences areprovided. Because of the source of the present invention, humans genomicsequence (FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleicacid molecules in the Figures will contain genomic intronic sequences,5′ and 3′ non-coding sequences, gene regulatory regions and non-codingintergenic sequences. In general such sequence features are either notedin FIGS. 1 and 3 or can readily be identified using computational toolsknown in the art. As discussed below, some of the non-coding regions,particularly gene regulatory elements such as promoters, are useful fora variety of purposes, e.g. control of heterologous gene expression,target for identifying gene activity modulating compounds, and areparticularly claimed as fragments of the genomic sequence providedherein.

[0113] The isolated nucleic acid molecules can encode the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

[0114] As mentioned above, the isolated nucleic acid molecules include,but are not limited to, the sequence encoding the kinase peptide alone,the sequence encoding the mature peptide and additional codingsequences, such as a leader or secretory sequence (e.g., a pre-pro orpro-protein sequence), the sequence encoding the mature peptide, with orwithout the additional coding sequences, plus additional non-codingsequences, for example introns and non-coding 5′ and 3′ sequences suchas transcribed but non-translated sequences that play a role intranscription, mRNA processing (including splicing and polyadenylationsignals), ribosome binding and stability of mRNA. In addition, thenucleic acid molecule may be fused to a marker sequence encoding, forexample, a peptide that facilitates purification.

[0115] Isolated nucleic acid molecules can be in the form of RNA, suchas mRNA, or in the form DNA, including cDNA and genomic DNA obtained bycloning or produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

[0116] The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the kinase proteins ofthe present invention that are described above. Such nucleic acidmolecules may be naturally occurring, such as allelic variants (samelocus), paralogs (different locus), and orthologs (different organism),or may be constructed by recombinant DNA methods or by chemicalsynthesis. Such non-naturally occurring variants may be made bymutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, as discussed above, thevariants can contain nucleotide substitutions, deletions, inversions andinsertions. Variation can occur in either or both the coding andnon-coding regions. The variations can produce both conservative andnon-conservative amino acid substitutions.

[0117] The present invention further provides non-coding fragments ofthe nucleic acid molecules provided in FIGS. 1 and 3. Preferrednon-coding fragments include, but are not limited to, promotersequences, enhancer sequences, gene modulating sequences and genetermination sequences. Such fragments are useful in controllingheterologous gene expression and in developing screens to identifygene-modulating agents. A promoter can readily be identified as being 5′to the ATG start site in the genomic sequence provided in FIG. 3.

[0118] A fragment comprises a contiguous nucleotide sequence greaterthan 12 or more nucleotides. Further, a fragment could at least 30, 40,50, 100, 250 or 500 nucleotides in length. The length of the fragmentwill be based on its intended use. For example, the fragment can encodeepitope bearing regions of the peptide, or can be useful as DNA probesand primers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

[0119] A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

[0120] Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene. As indicated by thedata presented in FIG. 3, the chromosome map position was determined tobe on human chromosome 6.

[0121]FIG. 3 provides information on a polymorphism (A738G) that wasidentified in the gene encoding the kinase proteins of the presentinvention.

[0122] As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45C, followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

[0123] Nucleic Acid Molecule Uses

[0124] The nucleic acid molecules of the present invention are usefulfor probes, primers, chemical intermediates, and in biological assays.The nucleic acid molecules are useful as a hybridization probe formessenger RNA, transcript/cDNA and genomic DNA to isolate full-lengthcDNA and genomic clones encoding the peptide described in FIG. 2 and toisolate cDNA and genomic clones that correspond to variants (alleles,orthologs, etc.) producing the same or related peptides shown in FIG. 2.As illustrated in FIG. 3, one SNP was identified (A738G).

[0125] The probe can correspond to any sequence along the entire lengthof the nucleic acid molecules provided in the Figures. Accordingly, itcould be derived from 5′ noncoding regions, the coding region, and 3′noncoding regions. However, as discussed, fragments are not to beconstrued as encompassing fragments disclosed prior to the presentinvention.

[0126] The nucleic acid molecules are also useful as primers for PCR toamplify any given region of a nucleic acid molecule and are useful tosynthesize antisense molecules of desired length and sequence.

[0127] The nucleic acid molecules are also useful for constructingrecombinant vectors. Such vectors include expression vectors thatexpress a portion of, or all of, the peptide sequences. Vectors alsoinclude insertion vectors, used to integrate into another nucleic acidmolecule sequence, such as into the cellular genome, to alter in situexpression of a gene and/or gene product. For example, an endogenouscoding sequence can be replaced via homologous recombination with all orpart of the coding region containing one or more specifically introducedmutations.

[0128] The nucleic acid molecules are also useful for expressingantigenic portions of the proteins.

[0129] The nucleic acid molecules are also useful as probes fordetermining the chromosomal positions of the nucleic acid molecules bymeans of in situ hybridization methods. As indicated by the datapresented in FIG. 3, the chromosome map position was determined to be onhuman chromosome 6.

[0130] The nucleic acid molecules are also useful in making vectorscontaining the gene regulatory regions of the nucleic acid molecules ofthe present invention.

[0131] The nucleic acid molecules are also useful for designingribozymes corresponding to all, or a part, of the mRNA produced from thenucleic acid molecules described herein.

[0132] The nucleic acid molecules are also useful for making vectorsthat express part, or all, of the peptides.

[0133] The nucleic acid molecules are also useful for constructing hostcells expressing a part, or all, of the nucleic acid molecules andpeptides.

[0134] The nucleic acid molecules are also useful for constructingtransgenic animals expressing all, or a part, of the nucleic acidmolecules and peptides.

[0135] The nucleic acid molecules are also useful as hybridizationprobes for determining the presence, level, form and distribution ofnucleic acid expression. Experimental data as provided in FIG. 1indicates that kinase proteins of the present invention are expressed inthe brain (hypothalamus and hippocampus), placenta, and mammary gland,as indicated by virtual northern blot analysis. Accordingly, the probescan be used to detect the presence of, or to determine levels of, aspecific nucleic acid molecule in cells, tissues, and in organisms. Thenucleic acid whose level is determined can be DNA or RNA. Accordingly,probes corresponding to the peptides described herein can be used toassess expression and/or gene copy number in a given cell, tissue, ororganism. These uses are relevant for diagnosis of disorders involvingan increase or decrease in kinase protein expression relative to normalresults.

[0136] In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA includes Southern hybridizations and in situhybridization.

[0137] Probes can be used as a part of a diagnostic test kit foridentifying cells or tissues that express a kinase protein, such as bymeasuring a level of a kinase-encoding nucleic acid in a sample of cellsfrom a subject e.g., mRNA or genomic DNA, or determining if a kinasegene has been mutated. Experimental data as provided in FIG. 1 indicatesthat kinase proteins of the present invention are expressed in the brain(hypothalamus and hippocampus), placenta, and mammary gland, asindicated by virtual northern blot analysis.

[0138] Nucleic acid expression assays are useful for drug screening toidentify compounds that modulate kinase nucleic acid expression.

[0139] The invention thus provides a method for identifying a compoundthat can be used to treat a disorder associated with nucleic acidexpression of the kinase gene, particularly biological and pathologicalprocesses that are mediated by the kinase in cells and tissues thatexpress it. Experimental data as provided in FIG. 1 indicates expressionin the brain (hypothalamus and hippocampus), placenta, and mammarygland. The method typically includes assaying the ability of thecompound to modulate the expression of the kinase nucleic acid and thusidentifing a compound that can be used to treat a disorder characterizedby undesired kinase nucleic acid expression. The assays can be performedin cell-based and cell-free systems. Cell-based assays include cellsnaturally expressing the kinase nucleic acid or recombinant cellsgenetically engineered to express specific nucleic acid sequences.

[0140] The assay for kinase nucleic acid expression can involve directassay of nucleic acid levels, such as mRNA levels, or on collateralcompounds involved in the signal pathway. Further, the expression ofgenes that are up- or down-regulated in response to the kinase proteinsignal pathway can also be assayed. In this embodiment the regulatoryregions of these genes can be operably linked to a reporter gene such asluciferase.

[0141] Thus, modulators of kinase gene expression can be identified in amethod wherein a cell is contacted with a candidate compound and theexpression of mRNA determined. The level of expression of kinase mRNA inthe presence of the candidate compound is compared to the level ofexpression of kinase mRNA in the absence of the candidate compound. Thecandidate compound can then be identified as a modulator of nucleic acidexpression based on this comparison and be used, for example to treat adisorder characterized by aberrant nucleic acid expression. Whenexpression of mRNA is statistically significantly greater in thepresence of the candidate compound than in its absence, the candidatecompound is identified as a stimulator of nucleic acid expression. Whennucleic acid expression is statistically significantly less in thepresence of the candidate compound than in its absence, the candidatecompound is identified as an inhibitor of nucleic acid expression.

[0142] The invention further provides methods of treatment, with thenucleic acid as a target, using a compound identified through drugscreening as a gene modulator to modulate kinase nucleic acid expressionin cells and tissues that express the kinase. Experimental data asprovided in FIG. 1 indicates that kinase proteins of the presentinvention are expressed in the brain (hypothalamus and hippocampus),placenta, and mammary gland, as indicated by virtual northern blotanalysis. Modulation includes both up-regulation (i.e. activation oragonization) or down-regulation (suppression or antagonization) ornucleic acid expression.

[0143] Alternatively, a modulator for kinase nucleic acid expression canbe a small molecule or drug identified using the screening assaysdescribed herein as long as the drug or small molecule inhibits thekinase nucleic acid expression in the cells and tissues that express theprotein. Experimental data as provided in FIG. 1 indicates expression inthe brain (hypothalamus and hippocampus), placenta, and mammary gland.

[0144] The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe kinase gene in clinical trials or in a treatment regimen. Thus, thegene expression pattern can serve as a barometer for the continuingeffectiveness of treatment with the compound, particularly withcompounds to which a patient can develop resistance. The gene expressionpattern can also serve as a marker indicative of a physiologicalresponse of the affected cells to the compound. Accordingly, suchmonitoring would allow either increased administration of the compoundor the administration of alternative compounds to which the patient hasnot become resistant. Similarly, if the level of nucleic acid expressionfalls below a desirable level, administration of the compound could becommensurately decreased.

[0145] The nucleic acid molecules are also useful in diagnostic assaysfor qualitative changes in kinase nucleic acid expression, andparticularly in qualitative changes that lead to pathology. The nucleicacid molecules can be used to detect mutations in kinase genes and geneexpression products such as mRNA. The nucleic acid molecules can be usedas hybridization probes to detect naturally occurring genetic mutationsin the kinase gene and thereby to determine whether a subject with themutation is at risk for a disorder caused by the mutation. Mutationsinclude deletion, addition, or substitution of one or more nucleotidesin the gene, chromosomal rearrangement, such as inversion ortransposition, modification of genomic DNA, such as aberrant methylationpatterns or changes in gene copy number, such as amplification.Detection of a mutated form of the kinase gene associated with adysfunction provides a diagnostic tool for an active disease orsusceptibility to disease when the disease results from overexpression,underexpression, or altered expression of a kinase protein.

[0146] Individuals carrying mutations in the kinase gene can be detectedat the nucleic acid level by a variety of techniques. FIG. 3 providesinformation on a polymorphism (A738G) that was identified in the geneencoding the kinase proteins of the present invention. As indicated bythe data presented in FIG. 3, the chromosome map position was determinedto be on human chromosome 6. Genomic DNA can be analyzed directly or canbe amplified by using PCR prior to analysis. RNA or cDNA can be used inthe same way. In some uses, detection of the mutation involves the useof a probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S.Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al., Science 241:1077-1080 (1988); and Nakazawa et al., PNAS91:360-364 (1994)), the latter of which can be particularly useful fordetecting point mutations in the gene (see Abravaya et al., NucleicAcids Res. 23:675-682 (1995)). This method can include the steps ofcollecting a sample of cells from a patient, isolating nucleic acid(e.g., genomic, mRNA or both) from the cells of the sample, contactingthe nucleic acid sample with one or more primers which specificallyhybridize to a gene under conditions such that hybridization andamplification of the gene (if present) occurs, and detecting thepresence or absence of an amplification product, or detecting the sizeof the amplification product and comparing the length to a controlsample. Deletions and insertions can be detected by a change in size ofthe amplified product compared to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to normal RNA orantisense DNA sequences.

[0147] Alternatively, mutations in a kinase gene can be directlyidentified, for example, by alterations in restriction enzyme digestionpatterns determined by gel electrophoresis.

[0148] Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site. Perfectly matchedsequences can be distinguished from mismatched sequences by nucleasecleavage digestion assays or by differences in melting temperature.

[0149] Sequence changes at specific locations can also be assessed bynuclease protection assays such as RNase and S1 protection or thechemical cleavage method. Furthermore, sequence differences between amutant kinase gene and a wild-type gene can be determined by direct DNAsequencing. A variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays (Naeve, C. W., (1995)Biotechniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

[0150] Other methods for detecting mutations in the gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242(1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth.Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant andwild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989);Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al.,Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant orwild-type fragments in polyacrylamide gels containing a gradient ofdenaturant is assayed using denaturing gradient gel electrophoresis(Myers et al., Nature 313:495 (1985)). Examples of other techniques fordetecting point mutations include selective oligonucleotidehybridization, selective amplification, and selective primer extension.

[0151] The nucleic acid molecules are also useful for testing anindividual for a genotype that while not necessarily causing thedisease, nevertheless affects the treatment modality. Thus, the nucleicacid molecules can be used to study the relationship between anindividual's genotype and the individual's response to a compound usedfor treatment (pharmacogenomic relationship). Accordingly, the nucleicacid molecules described herein can be used to assess the mutationcontent of the kinase gene in an individual in order to select anappropriate compound or dosage regimen for treatment. FIG. 3 providesinformation on a polymorphism (A738G) that was identified in the geneencoding the kinase proteins of the present invention.

[0152] Thus nucleic acid molecules displaying genetic variations thataffect treatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

[0153] The nucleic acid molecules are thus useful as antisenseconstructs to control kinase gene expression in cells, tissues, andorganisms. A DNA antisense nucleic acid molecule is designed to becomplementary to a region of the gene involved in transcription,preventing transcription and hence production of kinase protein. Anantisense RNA or DNA nucleic acid molecule would hybridize to the mRNAand thus block translation of mRNA into kinase protein.

[0154] Alternatively, a class of antisense molecules can be used toinactivate mRNA in order to decrease expression of kinase nucleic acid.Accordingly, these molecules can treat a disorder characterized byabnormal or undesired kinase nucleic acid expression. This techniqueinvolves cleavage by means of ribozymes containing nucleotide sequencescomplementary to one or more regions in the mRNA that attenuate theability of the mRNA to be translated. Possible regions include codingregions and particularly coding regions corresponding to the catalyticand other functional activities of the kinase protein, such as substratebinding.

[0155] The nucleic acid molecules also provide vectors for gene therapyin patients containing cells that are aberrant in kinase geneexpression. Thus, recombinant cells, which include the patient's cellsthat have been engineered ex vivo and returned to the patient, areintroduced into an individual where the cells produce the desired kinaseprotein to treat the individual.

[0156] The invention also encompasses kits for detecting the presence ofa kinase nucleic acid in a biological sample. Experimental data asprovided in FIG. 1 indicates that kinase proteins of the presentinvention are expressed in the brain (hypothalamus and hippocampus),placenta, and mammary gland, as indicated by virtual northern blotanalysis. For example, the kit can comprise reagents such as a labeledor labelable nucleic acid or agent capable of detecting kinase nucleicacid in a biological sample; means for determining the amount of kinasenucleic acid in the sample; and means for comparing the amount of kinasenucleic acid in the sample with a standard. The compound or agent can bepackaged in a suitable container. The kit can further compriseinstructions for using the kit to detect kinase protein mRNA or DNA.

[0157] Nucleic Acid Arrays

[0158] The present invention further provides nucleic acid detectionkits, such as arrays or microarrays of nucleic acid molecules that arebased on the sequence information provided in FIGS. 1 and 3 (SEQ IDNOS:1 and 3).

[0159] As used herein “Arrays” or “Microarrays” refers to an array ofdistinct polynucleotides or oligonucleotides synthesized on a substrate,such as paper, nylon or other type of membrane, filter, chip, glassslide, or any other suitable solid support. In one embodiment, themicroarray is prepared and used according to the methods described inU.S. Pat. No. 5,837,832, Chee et al., PCT application WO95/11995 (Cheeet al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) andSchena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all ofwhich are incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet al., U.S. Pat. No. 5,807,522.

[0160] The microarray or detection kit is preferably composed of a largenumber of unique, single-stranded nucleic acid sequences, usually eithersynthetic antisense oligonucleotides or fragments of cDNAs, fixed to asolid support. The oligonucleotides are preferably about 6-60nucleotides in length, more preferably 15-30 nucleotides in length, andmost preferably about 20-25 nucleotides in length. For a certain type ofmicroarray or detection kit, it may be preferable to useoligonucleotides that are only 7-20 nucleotides in length. Themicroarray or detection kit may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides which cover thefull length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray or detection kit may be oligonucleotides that arespecific to a gene or genes of interest.

[0161] In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the gene(s) of interest (or an ORFidentified from the contigs of the present invention) is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers are synthesized at designated areas on asubstrate using a light-directed chemical process. The substrate may bepaper, nylon or other type of membrane, filter, chip, glass slide or anyother suitable solid support.

[0162] In another aspect, an oligonucleotide may be synthesized on thesurface of the substrate by using a chemical coupling procedure and anink jet application apparatus, as described in PCT applicationWO95/251116 (Baldeschweiler et al.) which is incorporated herein in itsentirety by reference. In another aspect, a “gridded” array analogous toa dot (or slot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

[0163] In order to conduct sample analysis using a microarray ordetection kit, the RNA or DNA from a biological sample is made intohybridization probes. The mRNA is isolated, and cDNA is produced andused as a template to make antisense RNA (aRNA). The aRNA is amplifiedin the presence of fluorescent nucleotides, and labeled probes areincubated with the microarray or detection kit so that the probesequences hybridize to complementary oligonucleotides of the microarrayor detection kit. Incubation conditions are adjusted so thathybridization occurs with precise complementary matches or with variousdegrees of less complementarity. After removal of nonhybridized probes,a scanner is used to determine the levels and patterns of fluorescence.The scanned images are examined to determine degree of complementarityand the relative abundance of each oligonucleotide sequence on themicroarray or detection kit. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

[0164] Using such arrays, the present invention provides methods toidentify the expression of the kinase proteins/peptides of the presentinvention. In detail, such methods comprise incubating a test samplewith one or more nucleic acid molecules and assaying for binding of thenucleic acid molecule with components within the test sample. Suchassays will typically involve arrays comprising many genes, at least oneof which is a gene of the present invention and or alleles of the kinasegene of the present invention. FIG. 3 provides information on apolymorphism (A738G) that was identified in the gene encoding the kinaseproteins of the present invention.

[0165] Conditions for incubating a nucleic acid molecule with a testsample vary. Incubation conditions depend on the format employed in theassay, the detection methods employed, and the type and nature of thenucleic acid molecule used in the assay. One skilled in the art willrecognize that any one of the commonly available hybridization,amplification or array assay formats can readily be adapted to employthe novel fragments of the Human genome disclosed herein. Examples ofsuch assays can be found in Chard, T, An Introduction toRadioimmunoassay and Related Techniques, Elsevier Science Publishers,Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2(1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of EnzymeImmunoassays: Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

[0166] The test samples of the present invention include cells, proteinor membrane extracts of cells. The test sample used in theabove-described method will vary based on the assay format, nature ofthe detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing nucleic acid extracts or ofcells are well known in the art and can be readily be adapted in orderto obtain a sample that is compatible with the system utilized.

[0167] In another embodiment of the present invention, kits are providedwhich contain the necessary reagents to carry out the assays of thepresent invention.

[0168] Specifically, the invention provides a compartmentalized kit toreceive, in close confinement, one or more containers which comprises:(a) a first container comprising one of the nucleic acid molecules thatcan bind to a fragment of the Human genome disclosed herein; and (b) oneor more other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid.

[0169] In detail, a compartmentalized kit includes any kit in whichreagents are contained in separate containers. Such containers includesmall glass containers, plastic containers, strips of plastic, glass orpaper, or arraying material such as silica. Such containers allows oneto efficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified kinase gene of the present invention can beroutinely identified using the sequence information disclosed herein canbe readily incorporated into one of the established kit formats whichare well known in the art, particularly expression arrays.

[0170] Vectors/Host Cells

[0171] The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

[0172] A vector can be maintained in the host cell as anextrachromosomal element where it replicates and produces additionalcopies of the nucleic acid molecules. Alternatively, the vector mayintegrate into the host cell genome and produce additional copies of thenucleic acid molecules when the host cell replicates.

[0173] The invention provides vectors for the maintenance (cloningvectors) or vectors for expression (expression vectors) of the nucleicacid molecules. The vectors can function in prokaryotic or eukaryoticcells or in both (shuttle vectors).

[0174] Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

[0175] The regulatory sequence to which the nucleic acid moleculesdescribed herein can be operably linked include promoters for directingmRNA transcription. These include, but are not limited to, the leftpromoter from bacteriophage λ, the lac, TRP, and TAC promoters from E.coli, the early and late promoters from SV40, the CMV immediate earlypromoter, the adenovirus early and late promoters, and retroviruslong-terminal repeats.

[0176] In addition to control regions that promote transcription,expression vectors may also include regions that modulate transcription,such as repressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

[0177] In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual.2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

[0178] A variety of expression vectors can be used to express a nucleicacid molecule. Such vectors include chromosomal, episomal, andvirus-derived vectors, for example vectors derived from bacterialplasmids, from bacteriophage, from yeast episomes, from yeastchromosomal elements, including yeast artificial chromosomes, fromviruses such as baculoviruses, papovaviruses such as SV40, Vacciniaviruses, adenoviruses, poxviruses, pseudorabies viruses, andretroviruses. Vectors may also be derived from combinations of thesesources such as those derived from plasmid and bacteriophage geneticelements, e.g. cosmids and phagemids. Appropriate cloning and expressionvectors for prokaryotic and eukaryotic hosts are described in Sambrooket al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0179] The regulatory sequence may provide constitutive expression inone or more host cells (i.e. tissue specific) or may provide forinducible expression in one or more cell types such as by temperature,nutrient additive, or exogenous factor such as a hormone or otherligand. A variety of vectors providing for constitutive and inducibleexpression in prokaryotic and eukaryotic hosts are well known to thoseof ordinary skill in the art.

[0180] The nucleic acid molecules can be inserted into the vectornucleic acid by well-known methodology. Generally, the DNA sequence thatwill ultimately be expressed is joined to an expression vector bycleaving the DNA sequence and the expression vector with one or morerestriction enzymes and then ligating the fragments together. Proceduresfor restriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

[0181] The vector containing the appropriate nucleic acid molecule canbe introduced into an appropriate host cell for propagation orexpression using well-known techniques. Bacterial cells include, but arenot limited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

[0182] As described herein, it may be desirable to express the peptideas a fusion protein. Accordingly, the invention provides fusion vectorsthat allow for the production of the peptides. Fusion vectors canincrease the expression of a recombinant protein, increase thesolubility of the recombinant protein, and aid in the purification ofthe protein by acting for example as a ligand for affinity purification.A proteolytic cleavage site may be introduced at the junction of thefusion moiety so that the desired peptide can ultimately be separatedfrom the fusion moiety. Proteolytic enzymes include, but are not limitedto, factor Xa, thrombin, and enterokinase. Typical fusion expressionvectors include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.)which fuse glutathione S-transferase (GST), maltose E binding protein,or protein A, respectively, to the target recombinant protein. Examplesof suitable inducible non-fusion E. coli expression vectors include pTrc(Amann et al., Gene 69:301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185:60-89 (1990)).

[0183] Recombinant protein expression can be maximized in host bacteriaby providing a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990)119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

[0184] The nucleic acid molecules can also be expressed by expressionvectors that are operative in yeast. Examples of vectors for expressionin yeast e.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J.6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88(Schultz et al., Gene 54:113-123 (1987)), and pYES2 (InvitrogenCorporation, San Diego, Calif.).

[0185] The nucleic acid molecules can also be expressed in insect cellsusing, for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9cells) include the pAc series (Smith et al., Mol. Cell Biol. 3:2156-2165(1983)) and the pVL series (Lucklow et al., Virology 1 70:31-39 (1989)).

[0186] In certain embodiments of the invention, the nucleic acidmolecules described herein are expressed in mammalian cells usingmammalian expression vectors. Examples of mammalian expression vectorsinclude pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman etal., EMBO J. 6:187-195 (1987)).

[0187] The expression vectors listed herein are provided by way ofexample only of the well-known vectors available to those of ordinaryskill in the art that would be useful to express the nucleic acidmolecules. The person of ordinary skill in the art would be aware ofother vectors suitable for maintenance propagation or expression of thenucleic acid molecules described herein. These are found for example inSambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0188] The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

[0189] The invention also relates to recombinant host cells containingthe vectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

[0190] The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0191] Host cells can contain more than one vector. Thus, differentnucleotide sequences can be introduced on different vectors of the samecell. Similarly, the nucleic acid molecules can be introduced eitheralone or with other nucleic acid molecules that are not related to thenucleic acid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

[0192] In the case of bacteriophage and viral vectors, these can beintroduced into cells as packaged or encapsulated virus by standardprocedures for infection and transduction. Viral vectors can bereplication-competent or replication-defective. In the case in whichviral replication is defective, replication will occur in host cellsproviding functions that complement the defects.

[0193] Vectors generally include selectable markers that enable theselection of the subpopulation of cells that contain the recombinantvector constructs. The marker can be contained in the same vector thatcontains the nucleic acid molecules described herein or may be on aseparate vector. Markers include tetracycline or ampicillin-resistancegenes for prokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

[0194] While the mature proteins can be produced in bacteria, yeast,mammalian cells, and other cells under the control of the appropriateregulatory sequences, cell-free transcription and translation systemscan also be used to produce these proteins using RNA derived from theDNA constructs described herein.

[0195] Where secretion of the peptide is desired, which is difficult toachieve with multi-transmembrane domain containing proteins such askinases, appropriate secretion signals are incorporated into the vector.The signal sequence can be endogenous to the peptides or heterologous tothese peptides.

[0196] Where the peptide is not secreted into the medium, which istypically the case with kinases, the protein can be isolated from thehost cell by standard disruption procedures, including freeze thaw,sonication, mechanical disruption, use of lysing agents and the like.The peptide can then be recovered and purified by well-knownpurification methods including ammonium sulfate precipitation, acidextraction, anion or cationic exchange chromatography, phosphocellulosechromatography, hydrophobic-interaction chromatography, affinitychromatography, hydroxylapatite chromatography, lectin chromatography,or high performance liquid chromatography.

[0197] It is also understood that depending upon the host cell inrecombinant production of the peptides described herein, the peptidescan have various glycosylation patterns, depending upon the cell, ormaybe non-glycosylated as when produced in bacteria. In addition, thepeptides may include an initial modified methionine in some cases as aresult of a host-mediated process.

[0198] Uses of Vectors and Host Cells

[0199] The recombinant host cells expressing the peptides describedherein have a variety of uses. First, the cells are useful for producinga kinase protein or peptide that can be further purified to producedesired amounts of kinase protein or fragments. Thus, host cellscontaining expression vectors are useful for peptide production.

[0200] Host cells are also useful for conducting cell-based assaysinvolving the kinase protein or kinase protein fragments, such as thosedescribed above as well as other formats known in the art. Thus, arecombinant host cell expressing a native kinase protein is useful forassaying compounds that stimulate or inhibit kinase protein function.

[0201] Host cells are also useful for identifing kinase protein mutantsin which these functions are affected. If the mutants naturally occurand give rise to a pathology, host cells containing the mutations areuseful to assay compounds that have a desired effect on the mutantkinase protein (for example, stimulating or inhibiting function) whichmay not be indicated by their effect on the native kinase protein.

[0202] Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a kinase proteinand identifying and evaluating modulators of kinase protein activity.Other examples of transgenic animals include non-human primates, sheep,dogs, cows, goats, chickens, and amphibians.

[0203] A transgenic animal can be produced by introducing nucleic acidinto the male pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the kinase proteinnucleotide sequences can be introduced as a transgene into the genome ofa non-human animal, such as a mouse.

[0204] Any of the regulatory or other sequences useful in expressionvectors can form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the kinase protein to particularcells.

[0205] Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

[0206] In another embodiment, transgenic non-human animals can beproduced which contain selected systems that allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. PNAS89:6232-6236 (1992). Another example of a recombinase system is the FLPrecombinase system of S. cerevisiae (O'Gorman et al. Science251:1351-1355 (1991). If a cre/loxP recombinase system is used toregulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein is required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

[0207] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut, I. et al.Nature 385:810-813 (1997) and PCT International Publication Nos. WO97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growthcycle and enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

[0208] Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect substratebinding, kinase protein activation, and signal transduction, may not beevident from in vitro cell-free or cell-based assays. Accordingly, it isuseful to provide non-human transgenic animals to assay in vivo kinaseprotein function, including substrate interaction, the effect ofspecific mutant kinase proteins on kinase protein function and substrateinteraction, and the effect of chimeric kinase proteins. It is alsopossible to assess the effect of null mutations, that is, mutations thatsubstantially or completely eliminate one or more kinase proteinfunctions.

[0209] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of theabove-described modes for carrying out the invention which are obviousto those skilled in the field of molecular biology or related fields areintended to be within the scope of the following claims.

1 4 1 1338 DNA Homo sapiens 1 atgggggaga tgcagggcgc gctggccagagcccggctcg agtccctgct gcggccccgc 60 cacaaaaaga gggccgaggc gcagaaaaggagcgagtcct tcctgctgag cggactggct 120 ttcatgaagc agaggaggat gggtctgaacgactttattc agaagattgc caataactcc 180 tatgcatgca aacaccctga agttcagtccatcttgaaga tctcccaacc tcaggagcct 240 gagcttatga atgccaaccc ttctcctccaccaagtcctt ctcagcaaat caaccttggc 300 ccgtcgtcca atcctcatgc taaaccatctgactttcact tcttgaaagt gatcggaaag 360 ggcagttttg gaaaggttct tctagcaagacacaaggcag aagaagtgtt ctatgcagtc 420 aaagttttac agaagaaagc aatcctgaaaaagaaagagg agaagcatat tatgtcggag 480 cggaatgttc tgttgaagaa tgtgaagcaccctttcctgg tgggccttca cttctctttc 540 cagactgctg acaaattgta ctttgtcctagactacatta atggtggaga gttgttctac 600 catctccaga gggaacgctg cttcctggaaccacgggctc gtttctatgc tgctgaaata 660 gccagtgcct tgggctacct gcattcactgaacatcgttt atagagactt aaaaccagag 720 aatattttgc tagattcaca gggacacattgtccttactg acttcggact ctgcaaggag 780 aacattgaac acaacagcac aacatccaccttctgtggca cgccggagta tctcgcacct 840 gaggtgcttc ataagcagcc ttatgacaggactgtggact ggtggtgcct gggagctgtc 900 ttgtatgaga tgctgtatgg cctgccgcctttttatagcc gaaacacagc tgaaatgtac 960 gacaacattc tgaacaagcc tctccagctgaaaccaaata ttacaaattc cgcaagacac 1020 ctcctggagg gcctcctgca gaaggacaggacaaagcggc tcggggccaa ggatgacttc 1080 atggagatta agagtcatgt cttcttctccttaattaact gggatgatct cattaataag 1140 aagattactc ccccttttaa cccaaatgtgagtgggccca acgacctacg gcactttgac 1200 cccgagttta ccgaagagcc tgtccccaactccattggca agtcccctga cagcgtcctc 1260 gtcacagcca gcgtcaagga agctgccgaggctttcctag gcttttccta tgcgcctccc 1320 acggactctt tcctctga 1338 2 445 PRTHomo sapiens 2 Met Gly Glu Met Gln Gly Ala Leu Ala Arg Ala Arg Leu GluSer Leu 1 5 10 15 Leu Arg Pro Arg His Lys Lys Arg Ala Glu Ala Gln LysArg Ser Glu 20 25 30 Ser Phe Leu Leu Ser Gly Leu Ala Phe Met Lys Gln ArgArg Met Gly 35 40 45 Leu Asn Asp Phe Ile Gln Lys Ile Ala Asn Asn Ser TyrAla Cys Lys 50 55 60 His Pro Glu Val Gln Ser Ile Leu Lys Ile Ser Gln ProGln Glu Pro 65 70 75 80 Glu Leu Met Asn Ala Asn Pro Ser Pro Pro Pro SerPro Ser Gln Gln 85 90 95 Ile Asn Leu Gly Pro Ser Ser Asn Pro His Ala LysPro Ser Asp Phe 100 105 110 His Phe Leu Lys Val Ile Gly Lys Gly Ser PheGly Lys Val Leu Leu 115 120 125 Ala Arg His Lys Ala Glu Glu Val Phe TyrAla Val Lys Val Leu Gln 130 135 140 Lys Lys Ala Ile Leu Lys Lys Lys GluGlu Lys His Ile Met Ser Glu 145 150 155 160 Arg Asn Val Leu Leu Lys AsnVal Lys His Pro Phe Leu Val Gly Leu 165 170 175 His Phe Ser Phe Gln ThrAla Asp Lys Leu Tyr Phe Val Leu Asp Tyr 180 185 190 Ile Asn Gly Gly GluLeu Phe Tyr His Leu Gln Arg Glu Arg Cys Phe 195 200 205 Leu Glu Pro ArgAla Arg Phe Tyr Ala Ala Glu Ile Ala Ser Ala Leu 210 215 220 Gly Tyr LeuHis Ser Leu Asn Ile Val Tyr Arg Asp Leu Lys Pro Glu 225 230 235 240 AsnIle Leu Leu Asp Ser Gln Gly His Ile Val Leu Thr Asp Phe Gly 245 250 255Leu Cys Lys Glu Asn Ile Glu His Asn Ser Thr Thr Ser Thr Phe Cys 260 265270 Gly Thr Pro Glu Tyr Leu Ala Pro Glu Val Leu His Lys Gln Pro Tyr 275280 285 Asp Arg Thr Val Asp Trp Trp Cys Leu Gly Ala Val Leu Tyr Glu Met290 295 300 Leu Tyr Gly Leu Pro Pro Phe Tyr Ser Arg Asn Thr Ala Glu MetTyr 305 310 315 320 Asp Asn Ile Leu Asn Lys Pro Leu Gln Leu Lys Pro AsnIle Thr Asn 325 330 335 Ser Ala Arg His Leu Leu Glu Gly Leu Leu Gln LysAsp Arg Thr Lys 340 345 350 Arg Leu Gly Ala Lys Asp Asp Phe Met Glu IleLys Ser His Val Phe 355 360 365 Phe Ser Leu Ile Asn Trp Asp Asp Leu IleAsn Lys Lys Ile Thr Pro 370 375 380 Pro Phe Asn Pro Asn Val Ser Gly ProAsn Asp Leu Arg His Phe Asp 385 390 395 400 Pro Glu Phe Thr Glu Glu ProVal Pro Asn Ser Ile Gly Lys Ser Pro 405 410 415 Asp Ser Val Leu Val ThrAla Ser Val Lys Glu Ala Ala Glu Ala Phe 420 425 430 Leu Gly Phe Ser TyrAla Pro Pro Thr Asp Ser Phe Leu 435 440 445 3 10573 DNA Homo sapiens 3tctggctcgt gctctcatgt catctcagag ttccagctta tcagaggcat gtagcaggga 60ggcttattcc agccataact gggctctacc tccagcctcc agaagtaatc cccaacctgc 120atatccttgg gcaacccgaa gaatgaaaga agaagctata aaaccccctt tgaaaggttc 180gtacttaccg tactatattt tgcagatgcc tcaaaggatt tggggttact tggcatgggg 240aaggcacata aggtggggtg taggagaggg tctctggttg taggtttctt aatttaatgt 300ttgaaaacaa acatgcaaaa gtctgtgtgc aggttgatgt ttctgggcag cctgagcaaa 360atttgctctc tcaagaggga aaggaaccag gtgggagcag agctaggctg ggctaggcta 420gttgaatggt gggacatgac atacgggtgg cactggcaat aacaaagtca cattctatga 480agattccctg caagaggaag cagacatggg ccagttactg tgatttgaaa ttgcctaaac 540attgctttag gttggcatgt caatttcagg tactagtgtt ttttttgttt ttgtttttgt 600tttgtttttg tttgtttgtt tgttttgaga cggagtctcg ctctgttgcc aggctggagt 660gcagtggcgt gatctcggct cactgcaacc tccgcctccc gggttcaagc gattctcctg 720cctcagcctc ccgagtaact gggactacag gcgcacgcca ccacgcctgg ctaatttttc 780tattttcagt agagacgggg tttcaccatg ttagccagga tggtctcgat ctcttgacct 840cgtgatccgc ccgccttggc ctcccaaagt gctgggatta caggcgtgag ccactgcgcc 900cggccccagt aaatgctttt tataagtgtg ggcactgagc aaactttccc agccagactc 960caggagagag aatgtgtttc ccttctctcg gtttggggct gttgcaacaa agcaaaccaa 1020ggagttgaga ctagagctca ctttagggca agtgggggtg gttttgcctg caaaacaaac 1080ccctgcccaa gaccaaggaa aaggcgtttc acatgctatt cctggtttga cagctggtat 1140ttcgggactg tgccagatcc agtaggcaac tttaaaatgg cagagccttt ggtagcaaga 1200ggtcatggca gggcagccac cgcagacagc aacagcgagc gccaggtacc tggccctgcg 1260aatagtggta acttgtaact gcccgctccg ggcccagtcg ctgtgctcgc ggcttcccgg 1320ccagcactgg ctcacgtccc cgcgccggcg gtcaggctgc ggctcccaga catcccccag 1380ccgcggggtt actggaaggc accggcatcg ctgttctgca gagcccgggc cgccgcctcg 1440agcttccctc tcttccctgc cttctgcagc ggagtcaccc ggctaatctt tcaggataaa 1500gtcacagttt atgtgggact cacataaaga gcgagcgagg tggcaaaact aagaagccct 1560ggggcagcct tgagttaaac ccagggaggg tagggacgat tttaagacca tgtatcatga 1620cctgcagggt tttcaggtgg gacagcggga gaggagcagg ccccacagag gaatcgagga 1680tgcccggttc acgccaggtc tgcccccggg caaagctacc cctcccttcg cttgttacct 1740cctcacgtgt tcttggcatg gcagagatta aaaatgcaag gaaaaaaatt acatgcggaa 1800cggacaaaat gttctcagag attacttcag aaaaaaaaaa gtgaaatgca gattgtactt 1860cttcctttag tgcagagacg acttttattt ccgccccctc ccctccacat tcctgacctc 1920tccctccccc ttttccctct ttctttcctt ccttcctcct cttccaagtt ctgggatttt 1980tcagccttgc ttggttttgg ccaaaagcac aaaaaaggcg ttttcggaag cgacccgacc 2040gtgcacaagg gccatttgtt tgttttggga ctcggggcag gaaatcttgc ccggcctgag 2100tcacggcggc tccttcaagg aaacgtcagt gctcgccggt cgctctcgtc tgccgcgcgc 2160cccgccgccc gctgcccatg ggggagatgc agggcgcgct ggccagagcc cggctcgagt 2220ccctgctgcg gccccgccac aaaaagaggg ccgaggcgca gaaaaggagc gagtccttcc 2280tgctgagcgg actgggtaag cgccgccgcc ggccccgctg ggggcttggc tcacttcccc 2340agagcggctt ggaggcaggg gccggctttc gtcggagttc tcggggccgg ggtcccggcg 2400gcgggaacgg gaggacctgg cgggcgaggt cgcgcgcgca ggcctgcgcc ccagggataa 2460accccggagg gtggcgcgca ccgccggctc gggttgggga ggagggtggg agtccggccg 2520caggacggcg cctggccggg gagagggtat ctgcagggac agtgagcgaa gccaccgtgg 2580ccgccgcgca cccgccggga agcgcttcgg cgctgcgaac ccggctttct ccggcggcgg 2640aataaatgag agaggtggaa aactaccccg ggctctccgg ccctccccgc gccctccgcc 2700ggcgcgttct ctctctcctg ccccaggagc cgatggagac tgataacggc cctgcgccag 2760gccgtccccg ggcggtcctc gcgcccccgc ccggggctcg ccctctcaat ggggacagaa 2820ccgcccgccg caggcagcgt agccgccagc aaaccgcgag gcggtcgggg cggggcgagg 2880ggcgaggcga agggcggggc cacttctcac tgtcgcgcag gccccgcccc cgcggcggtg 2940ccttttttat aaggccgagc gcgcggcctg gcgcagcata cgccgagccg gtctttgagc 3000gctaacgtct ttctgtctcc ccgcggtggt gatgacggtg aaaactgagg ctgctaaggg 3060caccctcact tactccagga tgaggggcat ggtggcaatt ctcatcggtg agtgcaggaa 3120tcttgcggga cttctgctcc aggagacgca aagtggaaat tttttgaaag tcccggatca 3180gattagtgtg tgtggcgccg gacgttatga agccgtctaa acgtttcttt atttctcctc 3240cttcatccac agctttcatg aagcagagga ggatgggtct gaacgacttt attcagaaga 3300ttgccaataa ctcctatgca tgcaaacagt aagtttgacc ggatttgagg aaataactag 3360tatagtttga atttgccagc ggtaaacatt ctcatcacgg cgtttatcgg gaaggcgaag 3420acttcttctg gggtggggat ctcatttctc cttaaattct aatatatttg acacatttta 3480aacattaaag ttaatttgct gatttggctt gaactggaga tgtaagataa atggttcgtg 3540ttggccgaat tcacggcctt tctccatgag caacaatcct tatttctgta tttaatgggg 3600tttattattt tctttaactg actaatgtat tggggtattt tcagtttaaa cagtgaatta 3660tccgggtaga agtcggtaga gccaggaaac tcacttttga tgttggtgtg ccccctagtg 3720gcgagctgga ttctaaatcg tgccctttat tccctgcagc cctgaagttc agtccatctt 3780gaagatctcc caacctcagg agcctgagct tatgaatgcc aacccttctc ctccagtaag 3840tttttgtatg tgccgtgcat ctgtggagaa ctgtaaggga gtcagttagt attcctacat 3900taatggatta aaatagcatt tctagaaatt agtatcaagg caggaatgct tcattatggc 3960ataacaagtg atataaatat ttaagtattg agtcagagta ttattttatt tttttcctgg 4020gcatatttta cctccaaagt ggttatttta aaaggcatat ttcataaaaa ggttttatct 4080gtctgaaaca acatgactgt gtgcagtttc catactcatt tgaaatgtga tgaaatgtag 4140ttttgaatgt ttatagatgt atggtcattt gcatcagtca tttgtagatg taacattttc 4200tacatcgttt atgttataga tgtcttcctt tgaagcaatg gtattaaaag aaattctttt 4260tttttttttc tagccaagtc cttctcagca aatcaacctt ggcccgtcgt ccaatcctca 4320tgctaaacca tctgactttc acttcttgaa agtgatcgga aagggcagtt ttggaaaggt 4380aatttcaaat ctgaagatct tttggtacac ttccttcatg tcctctttta tattctccct 4440ggatgaggat agaaaaatga tttttttaaa ttgaaatttc aggttcttct agcaagacac 4500aaggcagaag aagtgttcta tgcagtcaaa gttttacaga agaaagcaat cctgaaaaag 4560aaagaggtat gagatgtgct tgatggggct ggcattggcg gtagacactc cttgaataat 4620cttgattctg gaatgttggt gccaagttga aacatgccac taaatctgaa tcgtcatttt 4680cctaggagaa gcatattatg tcggagcgga atgttctgtt gaagaatgtg aagcaccctt 4740tcctggtggg ccttcacttc tctttccaga ctgctgacaa attgtacttt gtcctagact 4800acattaatgg tggagaggtg agcagggggg atagaagtca actcttagtg tctctgcaca 4860gcctgctttg ttttagtttg agaaaaaagt tttcaaagat ttttggtggg gagaatgtta 4920ccagaattag catttccttc aacctgtcag gtttatagtt aatagattac ttggggccac 4980ttcctgcagt tgttcttttg ctgtgtatgt caaaactaat taaattcatt tgcaacccag 5040aatgactttg ttctgtctcc tgcagttgtt ctaccatctc cagagggaac gctgcttcct 5100ggaaccacgg gctcgtttct atgctgctga aatagccagt gccttgggct acctgcattc 5160actgaacatc gtttataggt aagcctgaga gctcttcagg ctaccagttt tggtataaag 5220gagacgtagc actggctgtt tcatagggcc ttaaaataat ttgtgtttat ttgcaacttg 5280gttgcctaaa accagatccc ctagcacgtg agctggcttg acttaagtgc caagggggaa 5340ccagccaagt aggattgtgc ctaatccaga atagatgagc agaacaaggg ctcccttttt 5400tcttcactac acaactacag tgaacctaaa atgcctctaa tacctttagc aattatcttt 5460aagaggatat cttatgaagt gaaattaact tgtgcaacta cttttctatt cactttttta 5520cagagactta aaaccagaga atattttgct agattcacag ggacacattg tccttactga 5580cttcggactc tgcaaggaga acattgaaca caacagcaca acatccacct tctgtggcac 5640gccggaggta ggcgctgtct tggtttggtg cctggtttac ccccgccttc caagagagag 5700atgtacaatc atgcacttaa ctaccaaaaa gagtaaactc ctctcagaga cttcttaata 5760cagttcagtg caaataaaat acatttgctg tttgatgtag catgagaaat cccaagtcct 5820tctgttcctt tactgaaaag tagctgtttg taagtaagat ctgcatcata aaaactttct 5880aaatccctaa gtaagagata tcaagtgccc agcagtttcc taaatgtcag tacacatagg 5940tagccagtca ccctcaaaaa gtccagcagt tttatcagga aggaatctaa agatatctat 6000cttccaagct ggctctgggt ctctcagctt tttcaaacta aatgtgtggt cgtgggattg 6060cttgctttcg caggttctaa acgctgtttc cctggtctgt ttttcagtat ctcgcacctg 6120aggtgcttca taagcagcct tatgacagga ctgtggactg gtggtgcctg ggagctgtct 6180tgtatgagat gctgtatggc ctggtgagtg gcacattggg aaccatggaa cactgcctgc 6240tccctacaat attgccttca cacagcccat gcttggccat ggtgtcttgc ccttaccagt 6300acgcttatca aaagcagcta agaggcatat tggttatttt atagttcata agaataatca 6360cttacctggt tcttttgtgc atttcacatt ttactagata ggaccacatt gaacctgtgt 6420ggtggtgaaa aactaccact tattaacatc taccccctca ccctccacac acacacacac 6480aaacacacac acgggttgca aagtagacac ttaaatagca agggaaaaga aagcattgag 6540gtggggagag tttctcaaat cgagcctaat atttattgcc gtttatatct ttttctctac 6600tggtaatgtg tgccatatga aacttccaat taagtctaaa gtaattttcc ccttctttca 6660gccgcctttt tatagccgaa acacagctga aatgtacgac aacattctga acaagcctct 6720ccagctgaaa ccaaatatta caaattccgc aagacacctc ctggagggcc tcctgcagaa 6780ggacaggaca aagcggctcg gggccaagga tgacttcgtg agtgatgttt tcctgtcctc 6840ctgggccggc cgggacgtgc actagacctc cctgccctta ttgaatgcac ctgtctaaat 6900taatcttggg tttcttatca acagatggag attaagagtc atgtcttctt ctccttaatt 6960aactgggatg atctcattaa taagaagatt actccccctt ttaacccaaa tgtggtgagt 7020atctgtctct cttctaagta tagagaagcc caaagggcat ttattttaat tcagaattgt 7080ctgggggagg gttggaagga atacattggc agatgttttc tccataaacc tgttatttta 7140cctacataaa aagcacattt ttgtgtccca acaaggctcc cataattttt agacacattt 7200atcaattcga agcaccaaaa ggcaacaagt gaacattatt cttatgttta actgtgtgta 7260gccttttgag attttgtgct tgaagtgggt gattatggaa gttgatataa gacttaaact 7320tggtatttaa agcctggtca agatttccct gtcctgtgtc tagtgtgagt tcttgacaag 7380agtgtttttc ccttcccgtc acagagtggg cccaacgacc tacggcactt tgaccccgag 7440tttaccgaag agcctgtccc caactccatt ggcaagtccc ctgacagcgt cctcgtcaca 7500gccagcgtca aggaagctgc cgaggctttc ctaggctttt cctatgcgcc tcccacggac 7560tctttcctct gaaccctgtt agggcttggt tttaaaggat tttatgtgtg tttccgaatg 7620ttttagttag ccttttggtg gagccgccag ctgacaggac atcttacaag agaatttgca 7680catctctgga agcttagcaa tcttattgca cactgttcgc tggaagcttt ttgaagagca 7740cattctcctc agtgagctca tgaggttttc atttttattc ttccttccaa cgtggtgcta 7800tctctgaaac gagcgttaga gtgccgcctt agacggaggc aggagtttcg ttagaaagcg 7860gacgctgttc taaaaaaggt ctcctgcaga tctgtctggg ctgtgatgac gaatattatg 7920aaatgtgcct tttctgaaga gattgtgtta gctccaaagc ttttcctatc gcagtgtttc 7980agttctttat tttcccttgt ggatatgctg tgtgaaccgt cgtgtgagtg tggtatgcct 8040gatcacagat ggattttgtt ataagcatca atgtgacact tgcaggacac tacaacgtgg 8100gacattgttt gtttcttcca tatttggaag ataaatttat gtgtagactt ttttgtaaga 8160tacggttaat aactaaaatt tattgaaatg gtcttgcaat gactcgtatt cagatgctta 8220aagaaagcat tgctgctaca aatatttcta tttttagaaa gggtttttat ggaccaatgc 8280cccagttgtc agtcagagcc gttggtgttt ttcattgttt aaaatgtcac ctgtaaaatg 8340ggcattattt atgttttttt ttttgcattc ctgataattg tatgtattgt ataaagaacg 8400tctgtacatt gggttataac actagtatat ttaaacttac aggcttattt gtaatgtaaa 8460ccaccatttt aatgtactgt aattaacatg gttataatac gtacaatcct tccctcatcc 8520catcacacaa ctttttttgt gtgtgataaa ctgattttgg tttgcaataa aaccttgaaa 8580aatatttaca tatattgtgt catgtgttat tttgtatatt ttggttaagg gggtaatcat 8640gggttagttt aaaattgaaa accatgaaaa tcctgctgta atttcctgct tagtggtttg 8700ctcccaacag cagtggtttc tgactccagg ggagtatagg atggtcttaa agccaaccta 8760cgttccaggc ctttttagca gcattttatg gtgtctgtca ttcataaatc catccaagga 8820aatcctttgc aatttactca tcttgcaagg attgctatga agtaatgctt cctgtattta 8880ttgcctgtcc tgtgaagttg gactatttgt cctgacattt ggcttgtctt cagttacagg 8940taattctttc cagaaatatt tgaaagccta ctctgggctc tattgcgagt gctcaggata 9000tcgtagtgga caaagcagac aacttcgccc ttccagagcc tgatgaagaa ggccgaccta 9060aagcagttag ttgagatgga aattgagaaa tagtctgtga agtttaggag aatgccacac 9120aagagggtga gaattttttt tttttttttt tttttttttg agacacggtc ttactctgtc 9180gcccaggctg gagtgcagtg gtgtgatctt ggttcactgc agcctccgcc tcctgggttc 9240atgtgatccc cccatctcag cctcctgagt agctgggact acaggcatgc accaccatgc 9300ctggctaatt tttgtatttt agtagagatg ggatttcacc atgtgggcca ggctggtctc 9360gaatccctgg cctcaagtga tctgtctgcc tcggcttccc taagtgctgg ggagaatgtt 9420ttaaataagt ggatatgttc ccaaaaagct gacctggctg ggacatctgg tttctgagag 9480tacctggagt tgacccaggt ctagagtgag ctcagtaaag ggaccctgaa ggagctcatc 9540cctagcttgg actgaagctt cttgagccag tgtctaccta gcaccctaag ggcccagcag 9600gctctggggc tgtgtggcag agcccactcc tagagctcac cccactgtga tattacctgt 9660gggagaaagc gaggtggcac catccttgga gatcttgagt ccaaaggttt ggactttttc 9720actcttctag gccttccaca caaatactta acaaataatc agggaatccc caaacagttg 9780atgttgctgc tgccttaatt gcaaaagcac cctgtaggcc tgctgcaccc ccgctaccct 9840gaccttccag ttcgcacagg gatttcccca agggaaagct gtgagctttt ttcctcttat 9900ccttgctctt gggtctcacc tcactttgcc tcagtccccc tctcctaccc cacaaggttt 9960ccaagggcca aacaggtgtt cagagataac cgagttcttc tccctcatga tctaatgaag 10020gaagaagatg aaaacgagtc gatagctttt tgctcaaggt gggccaccgg tcatgctctg 10080ctgttgactt actgctctac aggcattagc tacgtgttca attccctacc gggcccagtt 10140gacaaataaa gagtccaaag caaggccagg cacggtggct cacgcttgta atcccagcac 10200tttgggaggc cgaggcgggc agatcacgag gtcaggagat cgagaccatc ctggctaaca 10260tggtgaaacc ccgtctctac taaaaataca aaaaaattag ccgggcgtgg tggtgggcgc 10320ctgtagtccc agctactcgg gaggctgagg caggagaatg gcgtgaacca gggaggcgga 10380gcttgcagtg agccgagatc gcaccactgc actccagcct gggcgacaga gcaagactct 10440gtctcaaaaa acaaaacaaa acaaaagcat gtattttcct attaaagatt gatgccggct 10500ctaacataga gactcattgc atattccccc tcattctcat tctcaataac agttatgaat 10560tcctcctcga aca 10573 4 407 PRT Homo sapiens 4 Ile Ala Phe Met Lys GlnArg Arg Met Gly Leu Asn Asp Phe Ile Gln 1 5 10 15 Lys Ile Ala Asn AsnSer Tyr Ala Cys Lys His Pro Glu Val Gln Ser 20 25 30 Ile Leu Lys Ile SerGln Pro Gln Glu Pro Glu Leu Met Asn Ala Asn 35 40 45 Pro Ser Pro Pro ProSer Pro Ser Gln Gln Ile Asn Leu Gly Pro Ser 50 55 60 Ser Asn Pro His AlaLys Pro Ser Asp Phe His Phe Leu Lys Val Ile 65 70 75 80 Gly Lys Gly SerPhe Gly Lys Val Leu Leu Ala Arg His Lys Ala Glu 85 90 95 Glu Val Phe TyrAla Val Lys Val Leu Gln Lys Lys Ala Ile Leu Lys 100 105 110 Lys Lys GluGlu Lys His Ile Met Ser Glu Arg Asn Val Leu Leu Lys 115 120 125 Asn ValLys His Pro Phe Leu Val Gly Leu His Phe Ser Phe Gln Thr 130 135 140 AlaAsp Lys Leu Tyr Phe Val Leu Asp Tyr Ile Asn Gly Gly Glu Leu 145 150 155160 Phe Tyr His Leu Gln Arg Glu Arg Cys Phe Leu Glu Pro Arg Ala Arg 165170 175 Phe Tyr Ala Ala Glu Ile Ala Ser Ala Leu Gly Tyr Leu His Ser Leu180 185 190 Asn Ile Val Tyr Arg Asp Leu Lys Pro Glu Asn Ile Leu Leu AspSer 195 200 205 Gln Gly His Ile Val Leu Thr Asp Phe Gly Leu Cys Lys GluAsn Ile 210 215 220 Glu His Asn Ser Thr Thr Ser Thr Phe Cys Gly Thr ProGlu Tyr Leu 225 230 235 240 Ala Pro Glu Val Leu His Lys Gln Pro Tyr AspArg Thr Val Asp Trp 245 250 255 Trp Cys Leu Gly Ala Val Leu Tyr Glu MetLeu Tyr Gly Leu Pro Pro 260 265 270 Phe Tyr Ser Arg Asn Thr Ala Glu MetTyr Asp Asn Ile Leu Asn Lys 275 280 285 Pro Leu Gln Leu Lys Pro Asn IleThr Asn Ser Ala Arg His Leu Leu 290 295 300 Glu Gly Leu Leu Gln Lys AspArg Thr Lys Arg Leu Gly Ala Lys Asp 305 310 315 320 Asp Phe Met Glu IleLys Ser His Val Phe Phe Ser Leu Ile Asn Trp 325 330 335 Asp Asp Leu IleAsn Lys Lys Ile Thr Pro Pro Phe Asn Pro Asn Val 340 345 350 Ser Gly ProAsn Asp Leu Arg His Phe Asp Pro Glu Phe Thr Glu Glu 355 360 365 Pro ValPro Asn Ser Ile Gly Lys Ser Pro Asp Ser Val Leu Val Thr 370 375 380 AlaSer Val Lys Glu Ala Ala Glu Ala Phe Leu Gly Phe Ser Tyr Ala 385 390 395400 Pro Pro Thr Asp Ser Phe Leu 405

That which is claimed is:
 1. An isolated peptide consisting of an aminoacid sequence selected from the group consisting of: (a) an amino acidsequence shown in SEQ ID NO: 2; (b) an amino acid sequence of an allelicvariant of an amino acid sequence shown in SEQ ID NO: 2, wherein saidallelic variant is encoded by a nucleic acid molecule that hybridizesunder stringent conditions to the opposite strand of a nucleic acidmolecule shown in SEQ ID NOS: 1 or 3; (c) an amino acid sequence of anortholog of an amino acid sequence shown in SEQ ID NO: 2, wherein saidortholog is encoded by a nucleic acid molecule that hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS: 1 or 3; and (d) a fragment of an amino acidsequence shown in SEQ ID NO: 2, wherein said fragment comprises at least10 contiguous amino acids.
 2. An isolated peptide comprising an aminoacid sequence selected from the group consisting of: (a) an amino acidsequence shown in SEQ ID NO: 2; (b) an amino acid sequence of an allelicvariant of an amino acid sequence shown in SEQ ID NO: 2, wherein saidallelic variant is encoded by a nucleic acid molecule that hybridizesunder stringent conditions to the opposite strand of a nucleic acidmolecule shown in SEQ ID NOS: 1 or 3; (c) an amino acid sequence of anortholog of an amino acid sequence shown in SEQ ID NO: 2, wherein saidortholog is encoded by a nucleic acid molecule that hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS: 1 or 3; and (d) a fragment of an amino acidsequence shown in SEQ ID NO: 2, wherein said fragment comprises at least10 contiguous amino acids.
 3. An isolated antibody that selectivelybinds to a peptide of claim
 2. 4. An isolated nucleic acid moleculeconsisting of a nucleotide sequence selected from the group consistingof: (a) a nucleotide sequence that encodes an amino acid sequence shownin SEQ ID NO: 2; (b) a nucleotide sequence that encodes of an allelicvariant of an amino acid sequence shown in SEQ ID NO: 2, wherein saidnucleotide sequence hybridizes under stringent conditions to theopposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3;(c) a nucleotide sequence that encodes an ortholog of an amino acidsequence shown in SEQ ID NO: 2, wherein said nucleotide sequencehybridizes under stringent conditions to the opposite strand of anucleic acid molecule shown in SEQ ID NOS: 1 or 3; (d) a nucleotidesequence that encodes a fragment of an amino acid sequence shown in SEQID NO: 2, wherein said fragment comprises at least 10 contiguous aminoacids; and (e) a nucleotide sequence that is the complement of anucleotide sequence of (a)-(d).
 5. An isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:(a) a nucleotide sequence that encodes an amino acid sequence shown inSEQ ID NO: 2; (b) a nucleotide sequence that encodes of an allelicvariant of an amino acid sequence shown in SEQ ID NO: 2, wherein saidnucleotide sequence hybridizes under stringent conditions to theopposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3;(c) a nucleotide sequence that encodes an ortholog of an amino acidsequence shown in SEQ ID NO: 2, wherein said nucleotide sequencehybridizes under stringent conditions to the opposite strand of anucleic acid molecule shown in SEQ ID NOS: 1 or 3; (d) a nucleotidesequence that encodes a fragment of an amino acid sequence shown in SEQID NO: 2, wherein said fragment comprises at least 10 contiguous aminoacids; and (e) a nucleotide sequence that is the complement of anucleotide sequence of (a)-(d).
 6. A gene chip comprising a nucleic acidmolecule of claim
 5. 7. A transgenic non-human animal comprising anucleic acid molecule of claim
 5. 8. A nucleic acid vector comprising anucleic acid molecule of claim
 5. 9. A host cell containing the vectorof claim
 8. 10. A method for producing any of the peptides of claim 1comprising introducing a nucleotide sequence encoding any of the aminoacid sequences in (a)-(d) into a host cell, and culturing the host cellunder conditions in which the peptides are expressed from the nucleotidesequence.
 11. A method for producing any of the peptides of claim 2comprising introducing a nucleotide sequence encoding any of the aminoacid sequences in (a)-(d) into a host cell, and culturing the host cellunder conditions in which the peptides are expressed from the nucleotidesequence.
 12. A method for detecting the presence of any of the peptidesof claim 2 in a sample, said method comprising contacting said samplewith a detection agent that specifically allows detection of thepresence of the peptide in the sample and then detecting the presence ofthe peptide.
 13. A method for detecting the presence of a nucleic acidmolecule of claim 5 in a sample, said method comprising contacting thesample with an oligonucleotide that hybridizes to said nucleic acidmolecule under stringent conditions and determining whether theoligonucleotide binds to said nucleic acid molecule in the sample.
 14. Amethod for identifying a modulator of a peptide of claim 2, said methodcomprising contacting said peptide with an agent and determining if saidagent has modulated the function or activity of said peptide.
 15. Themethod of claim 14, wherein said agent is administered to a host cellcomprising an expression vector that expresses said peptide.
 16. Amethod for identifying an agent that binds to any of the peptides ofclaim 2, said method comprising contacting the peptide with an agent andassaying the contacted mixture to determine whether a complex is formedwith the agent bound to the peptide.
 17. A pharmaceutical compositioncomprising an agent identified by the method of claim 16 and apharmaceutically acceptable carrier therefor.
 18. A method for treatinga disease or condition mediated by a human kinase protein, said methodcomprising administering to a patient a pharmaceutically effectiveamount of an agent identified by the method of claim
 16. 19. A methodfor identifying a modulator of the expression of a peptide of claim 2,said method comprising contacting a cell expressing said peptide with anagent, and determining if said agent has modulated the expression ofsaid peptide.
 20. An isolated human kinase peptide having an amino acidsequence that shares at least 70% homology with an amino acid sequenceshown in SEQ ID NO:
 2. 21. A peptide according to claim 20 that sharesat least 90 percent homology with an amino acid sequence shown in SEQ IDNO:
 2. 22. An isolated nucleic acid molecule encoding a human kinasepeptide, said nucleic acid molecule sharing at least 80 percent homologywith a nucleic acid molecule shown in SEQ ID NOS: 1 or
 3. 23. A nucleicacid molecule according to claim 22 that shares at least 90 percenthomology with a nucleic acid molecule shown in SEQ ID NOS: 1 or 3.